Study of cationic polymer P(DMA⁺-s-HEMA) prepared via ATRP method for protein immobilisation

The surface design of the interface affects the performance of protein immobilisation. Hence, a well-controlled polymer surface functional group should take place to improve the protein immobilisation efficiency. Thus, this study aimed to synthesise cationic polymer, poly(2-(N,N-dimethylamino)ethylmethacrylate-stat-2-hydroxyethyl methacrylate (P(DMA+-s-HEMA)) via Atom Transfer Radical Polymerisation (ATRP) method to produce more defined polymers. Prior to the P(DMA+-s-HEMA) study, a preliminary study on the stability of the model enzyme horseradish peroxidase (HRP) was conducted. The preliminary study aims to investigate the effect of methyl methacrylate (MMA) to methacrylic acid (MAA) feed ratio on the shape, morphology and average diameter (Dave) of microparticles (MPs) on protein immobilisation. MPs were prepared using the solvent evaporation method with four different parameters: polymer types (PMMA98 and PMMA80), the molecular weight of the polyvinyl alcohol, (PVA (PVA13K and PVA130K)), surfactant types (PVA and poly(vinyl pyrrolidone) (PVP)), and volume fraction of oil phase (φo= 0.30, 0.50 and 0.70). Furthermore, HRP was then immobilised onto MPs and the stability study was measured for seven days. All immobilised HRP showed relatively stable activity, with the highest relative activity percentage recorded by the PMMA98/PVP system (67.5%), suggesting this MPs is the most suitable for enzyme immobilisation among the other MPs produced. The preliminary study concluded that MMA to MAA feed ratio, morphology and Dave of the MPs affect the stability of HRP. Then, the main part of the study focused on the synthesis of P(DMA⁺-s-HEMA) prepared via the ATRP method for bioassay application, investigating the effects of two different molar ratios of dimethylaminoethyl methacrylate to 2-hydroxyl methacrylate ([DMA]:[HEMA]=1:1 and 3:1) and its size (Dh) on protein immobilisation. The NMR spectroscopy shows the P(DMA+-s-HEMA) was successfully synthesised and quaternised. The average zeta potential (ζ) values increase from 12 to 62 mV when the [DMA] ratio increases, indicating that they can adhere to the negatively charged substrate (quartz) using a simple Chemical Bath Deposition (CBD) method to produce thin films, with pendant hydroxyl groups that were linked to a model protein, Concanavalin A (Con A) via carbonyldiimidazole (CDI) chemistry. Atomic Force Microscopy (AFM) analyses show that the L3⁺:1 thin film and its Con A immobilisation have the highest surface roughness, indicating an increase in the [DMA]:[HEMA] and its appropriate size may lead to better protein immobilisation. The results showed that the molar ratio of 3:1 was the most suitable for protein immobilisation. In conclusion, this study highlights the importance of functional group ratios, surface morphology, and Dh affecting protein immobilisation in bioassay. Overall, our study shows that P(DMA⁺-s-HEMA) prepared using the ATRP method with a controlled concentration of hydroxyl groups can be used for high-density protein immobilisation and has a potential for bioassay for future biosensor development.