A high-throughput integrated biofilm-on-a-chip platform for the investigation of combinatory physicochemical responses to chemical and fluid shear stress

Abstract

Physicochemical conditions play a key role in the development of biofilm removal strategies. This study presents an integrated, double-layer, high-throughput microfluidic chip for real-time screening of the combined effect of antibiotic concentration and fluid shear stress (FSS) on biofilms. Biofilms of Escherichia coli LF82 and Pseudomonas aeruginosa were tested against gentamicin and streptomycin to examine the time dependent effects of concentration and FSS on the integrity of the biofilm. A MatLab image analysis method was developed to measure the bacterial surface coverage and total fluorescent intensity of the biofilms before and after each treatment. The chip consists of two layers. The top layer contains the concentration gradient generator (CGG) capable of diluting the input drug linearly into four concentrations. The bottom layer contains four expanding FSS chambers imposing three different FSSs on cultured biofilms. As a result, 12 combinatorial states of concentration and FSS can be investigated on the biofilm simultaneously. Our proof-of-concept study revealed that the reduction of E. coli biofilms was directly dependent upon both antibacterial dose and shear intensity, whereas the P. aeruginosa biofilms were not impacted as significantly. This confirmed that the effectiveness of biofilm removal is dependent on bacterial species and the environment. Our experimental system could be used to investigate the physicochemical responses of other biofilms or to assess the effectiveness of biofilm removal methods.

Fig 1. Integrated microfluidic platform allows investigation of combinatory effects of chemical and fluid shear stress (FSS).

Ports and channels on the top layer are colored in red. Microchambers and collector channels on the bottom layer are colored in green. Vertical cylinders mediate the connection between the channels on the top and the bottom layer A) Schematic representation of the 2-layer physicochemical analysis biofilmchip (2PAB) B) Cross-sectional view of the platform with red arrows represent schematically the fluid flow streamline, and detailed dimensions of key features of the FSS chambers. C) Detailed schematic of the device with the concentration gradient generator (CGG) on the top layer providing stepwise antibiotic dilutions, and the FSS chambers on the bottom layer providing three zones of different shear stress levels in each chamber for testing on established biofilms. D) The workflow for biofilm formation and antibiotic/FSS treatment. Red arrows indicate the direction of fluid flow when applicable.

Fig 2. Characterization of concentration gradient formation.

(A) Computational fluid dynamic (CFD) simulation of the concentration gradient generator (CGG) and (B) experimentally obtained fluorescent images of the resulting concentration gradient. Graph showing normalized concentrations of resazurin in four FSS chambers. FSS chambers 1 and 4 were set as 0 and 100 relative concentrations, and the mean relative concentration ± standard deviation of FSS chamber 2 and 3 are shown.

Fig 3. Characterization of FSS distribution on cultured biofilms for each of the three zones of the expanding chamber by computational fluid dynamic simulation.

Except for a small fraction of the surface near the transitions between the zones, the FSS is uniform along the expanding chambers on the biofilm.

Fig 4

A) Fluorescent images of the on-chip E. coli LF82 biofilms and the physicochemical effects of increasing concentration of gentamicin and fluid shear stress after 24 h treatment. B) Relative changes in surface coverage and total fluorescent intensity of E. coli biofilms after 24 h treatment under different concentrations of gentamicin and levels of fluid shear stress were evaluated by MatLab analysis of fluorescent images. Data represent the mean of three replicates, reported as the percentage normalized with the initial time point (T₀) of each treatment. Values are expressed as means ± SEM, n = 3. Scale bar = 100μm.