Life under flow: A novel microfluidic device for the assessment of anti-biofilm technologies

Abstract

In the current study, we have developed and fabricated a novel lab-on-a-chip device for the investigation of biofilm responses, such as attachment kinetics and initial biofilm formation, to different hydrodynamic conditions. The microfluidic flow channels are designed using computational fluid dynamic simulations so as to have a pre-defined, homogeneous wall shear stress in the channels, ranging from 0.03 to 4.30 Pa, which are relevant to in-service conditions on a ship hull, as well as other man-made marine platforms. Temporal variations of biofilm formation in the microfluidic device were assessed using time-lapse microscopy, nucleic acid staining, and confocal laser scanning microscopy (CLSM). Differences in attachment kinetics were observed with increasing shear stress, i.e., with increasing shear stress there appeared to be a delay in bacterial attachment, i.e., at 55, 120, 150, and 155 min for 0.03, 0.60, 2.15, and 4.30 Pa, respectively. CLSM confirmed marked variations in colony architecture, i.e.,: (i) lower shear stresses resulted in biofilms with distinctive morphologies mainly characterised by mushroom-like structures, interstitial channels, and internal voids, and (ii) for the higher shear stresses compact clusters with large interspaces between them were formed. The key advantage of the developed microfluidic device is the combination of three architectural features in one device, i.e., an open-system design, channel replication, and multiple fully developed shear stresses.

Figure 1

The workflow for the μFD design optimisation.

Figure 2

The μFD used to assess bacterial attachment. The cross section (left) and top view (right) of the four parts of the assembly are shown together with their dimensions where (a) top plate, (b) gasket, (c) experimental substrate (in this case glass), (d) bottom plate, and (e) cross-section of an individual channel with all the chambers (this is replicated 6times).

Figure 3

Schematic illustrating the μFD system, closed-loop, microscope set-up and a close-up of the μFD. Arrows indicate the flow direction.

Figure 4

(a) Cross-section schematic of the micro-channel showing the dimensions of the four micro-chambers. The flow field modelling within the channel, using a flow rate of 0.5 ml min⁻¹ at the inlet, (b) top-view colour map of the modelled wall shear stress on the substrate surface, (c) the wall shear stress along the cross section has been determined at the middle length of each chamber section, (d) wall shear stress at different positions along the channel, computed at half-width length of the channel sections showing the characteristic stairlike increase of the wall shear stress.

Figure 5

Hydrodynamic performance of C. marina attachment and initial biofilm formation (percentage of surface coverage) with time in single chambers under (a) 0.03 Pa, (b) 0.60 Pa, (c) 2.15 Pa, and (d) 4.30 Pa, Error bars ± SD. Figure (e) shows the C. marina's surface coverage (%) at two time points, i.e., start of the experiment (t₃₀) and at the end point (t₂₁₀) for all channels (N = 48). Error bars ± SE. All data were acquired under flow conditions and at the centre of each chamber.

Figure 6

Representative CLSM images of C. marina stained with SYTO9 and PI, grown in the μFD and exposed under different shear stresses at the end-point (t₂₁₀), where Top: 0.03 Pa, Bottom: 0.60 Pa and (a) top view, (b) side view, and (c) 3D view. Each grid square is equivalent to 38 μm², scale bars: 100 μm; white arrows illustrate the direction of the flow.

Figure 7

Representative CLSM images of C. marina stained with SYTO9 and PI, grown in the μFD and exposed under different shear stresses at the end-point (t₂₁₀), where Top: 2.15 Pa, Bottom: 4.30 Pa and (a) top view, (b) side view, and (c) 3D view. Each grid square is equivalent to 38 μm², scale bars: 100 μm; white arrows illustrate the direction of the flow.

Figure 8

Representative intensity map reflecting biofilm thickness (scale in μm) grown in the μFD at the end-point. Each image represents a flow chamber for each shear stress, i.e., (a) 0.03 Pa, (b) 0.60 Pa, (c) 2.15 Pa, and (c) 4.30 Pa.

Figure 9

Representative profiles of bacterial vertical distribution from the glass surface to the fluid phase determined from 3D CLSM imaging data sets of newly formed biofilms grown in the μFD and under four different shear stresses, i.e., (a) 0.03 and 0.60 Pa and (b) 2.15 and 4.30 Pa.