Horseradish peroxidase-catalyzed polyacrylamide gels: monitoring their polymerization with BSA-stabilized gold nanoclusters and their functional validation in electrophoresis

Abstract

Polyacrylamide gel (PAG) is extensively used as a matrix for biomolecular analysis and fractionation. However, the traditional polymerization catalyst system N,N,N′,N′-tetramethylethylenediamine (TEMED)/ammonium persulphate (APS) of PAG presents non-negligible toxicity. Herein, we utilized the green and efficient bio-enzyme horseradish peroxidase (HRP) to catalyze the gel polymerization of polyacrylamide. At the same time, the efficacy of this gel system in separating nucleic acids and proteins was confirmed by applying the gel system in electrophoresis. This study aims to explore a higher biosafety polyacrylamide gel polymerization catalytic system which can be applied to electrophoresis technology. Furthermore, in order to differentiate between the bio-enzymatic catalytic system and the traditional toxic catalytic system during polymerization, aggregation-induced luminescence (AIE) of bovine serum albumin-stabilized gold nanoclusters (BSA-Au NCs) was used to monitor the polymerization reaction of the system. The results indicated that the fluorescence intensity of the polymeric system containing BSA-Au NCs increased with the polymerization of the monomers. Subsequently, we assessed whether certain components of nucleic acid electrophoresis and protein electrophoresis such as sodiumdodecylsulfate (SDS) and TBE buffer (Tris-boric acid, EDTA, pH 8.3) would affect the polymerization of the polyacrylamide gels catalyzed by the biological enzymes. The experimental conditions were also optimized to explore the optimal concentration of the ternary system of HRP, H₂O₂ and ACAC. Our results suggested that the bioenzyme-catalyzed system could be a feasible alternative to the TEMED/APS-catalyzed system, which also could provide new insights into the methods of monitoring the polymerization system.