Lysis of gram-positive and gram-negative bacteria by antibacterial porous polymeric monolith formed in microfluidic biochips for sample preparation

Abstract

Bacterial cell lysis is demonstrated using polymeric microfluidic biochips operating via a hybrid mechanical shearing/contact-killing mechanism. These biochips are fabricated from a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by well-controlled, high-throughput laser micromachining. The unreacted double bonds at the surface of X-PMMA provide covalent bonding for the monolith, thus contributing to the mechanical stability of the biochip and eliminating the need for surface treatment. The lysis efficiency of these biochips was tested for gram-positive (Enterococcus saccharolyticus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Pseudomonas fluorescens), and confirmed by off-chip PCR without further purification. The influence of the flow-rate when pumping the bacterial suspension through the monolith, and of the hydrophobic-hydrophilic balance on the cell lysis efficiency was investigated at a cell concentration of 10 5 CFU/ml. The lysis efficiency of the biochips was better than for off-chip chemical, mechanical, and thermal lysis techniques. The biochip also acts as a filter that isolates cell debris and allows PCR-amplifiable DNA to pass through. As expected, the system performs more efficient lysis for gram-negative than for gram-positive bacteria. The biochip does not require chemical/enzymatic reagents, power consumption, or complicated design and fabrication processes, which makes it an attractive on-chip lysis device that can be used in sample preparation for bio-genetics and point-of-care diagnostics. The biochips were reused for 20 lysis cycles without significant performance degradation or carryover when they were back-flushed between cycles. The biochips efficiently lysed both gram-positive and gram-negative bacteria in about 35 min per lysis and monolith regeneration cycle.

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

“…The antimicrobial polymers have the ability to inhibit microbial organisms by killing them. The same group later improved the efficiency of lysis (89%) by regulating the hydrophobic–hydrophilic property of the PPM [ 50 ]. They also suggest that use of PPM did not inhibit the PCR amplification process.…”

Advances in monoliths and related porous materials for microfluidics

“…These monoliths allowed specific capture of Rickettsia typhi, and subsequent immunofluorescent staining and imaging of the bacteria inside the chip indicated detection limits of about 100 bacteria per ml of blood sample. Aly et al 90,91 developed a method for lysis of both gram-positive and gram-negative bacteria using a monolith based on in-house-synthesized N-(tert-butyloxycarbonyl) aminoethyl methacrylate, which has antibacterial properties. The influence of the flow rate and of the hydrophilic-lipophilic balance in the monolith on the cell lysis efficiency was investigated, and they found that the contribution of contact killing to cell lysis was more significant than that of mechanical shearing.…”

A High-Performance Antibacterial Nanostructured ZnO Microfluidic Device for Controlled Bacterial Lysis and DNA Release

“…The development of antibacterial materials with nanostructured features has shown promising results for reducing the adhesion on surfaces [ 17 , 18 ] or even killing bacteria due to the fact that cells respond to nanostructures of specific topography by deforming or extended stretching of their membrane [ 19 , 20 , 21 ], resulting to cell piercing or rupture [ 22 , 23 , 24 , 25 , 26 ]. The most well-known materials with active antibacterial properties are nanosized metal oxide compounds and nanostructures thereof (TiO 2 , ZnO, CuO) which are exploited as antimicrobial surfaces [ 19 , 27 , 28 ], as well as in microfluidic and highly efficient analytical platforms [ 29 ].…”