Antibacterial porous polymeric monolith columns with amphiphilic and polycationic character on cross-linked PMMA substrates for cell lysis applications

Abstract

The application of porous polymeric monolith (PPM) columns as an effective tool for bacterial cell lysis within microfluidic chips is demonstrated. By taking advantage of the large surface area and controllable pore size inherent to PPMs, we developed a double mechanism cell lysis technique. The bacterial cell wall is mechanically sheared by flowing through the porous medium of the PPM column, but it is also damaged and disintegrated by physical contact with the antibacterial polymeric biocide covering the porous surface. This leads to leakage of the intracellular contents. The stable and non-leaching antibacterial column introduced in this work alleviates the need for chemical or enzymatic lysins and their potential release of polymerase chain reaction (PCR) inhibitors. The PPM columns were obtained by the photoinitiated free radical copolymerization of n-butyl methacrylate (BuMA) and N-(tert-butyloxycarbonyl)aminoethyl methacrylate (Boc-AEMA) in the presence of a cross-linker and porogenic solvents. The porous network was synthesized directly inside a microfluidic channel fabricated in a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by laser micromachining. After removing the Boc protecting group with phosphoric acid, an amphiphilic and cationic network structure reminiscent of synthetic mimics of antimicrobial peptides (SMAMPs) was obtained. The antibacterial activity of the PPM columns was tested against Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli) cells. Cell lysis was evidenced by DNA release, which was then amplified by PCR and confirmed by gel electrophoresis, to verify that the antibacterial monolithic columns did not strongly interfere with the PCR process.