Centrifugal microfluidic platform for optical monitoring of bacterial biofilms

Most bacteria can adhere to various surfaces, included human tissues, developing a complex 3D structure called biofilm[5], which has high resistance to disinfectant chemicals and antibiotics[6].Commonly in hospitals, the analysis of antibiotics resistance is carried out using static bacterial cultures in Petri dishes, which are time and resource consuming and then they do not mimic the in vivo like conditions.In the last few decades in research laboratories, bacterial cultures are also carried out in microfluidic systems where bacterias are cultured in flow fluids mimicking, in this way, more in vivo like conditions. However, most of the pumps used to move liquid in the microfluidic systems are complex and bulky, often requiring large amount of reagents and culture medium[7].Centrifugal microfluidic platforms, “also referred to as lab-on-a-disc (LOD)”, have a simple fluid actuation module to pump and handle the fluid inside the platforms. These systems require a simple motor to rotate the disc in order to exploit the forces which arise from rotation to manipulate the fluid [8]. These systems without auxiliary pumps and tubing require low sample volumes and could potentially decrease of the possibility of contaminations.The aim of this project was to create a LOD system which was enabled low flow rates (few hundreds nl/min), suitable for perfusion culture. The system was fabricated in Polycarbonate (PC), which is biocompatible material and can resist the commonly used sterilization methods. The final disc was composed of two PC layers. One layer presented the inlet, outlet reservoirs, cell culture chamber and micro-channels and the second layer, containing the inlet and venting holes, was used as a lid to have a closed system. The structures in bottom layer were fabricated with micromilling, while the inlet and venting holes on the lid were done with a drill. The two layers were bonded with thermal bonding. The flow rate were measured optically and calculated with an image analysis through a Matlab code. The flow rate were evaluated at different rotational frequencies ranged from 1.125 Hz to 0.375 Hz.The lowest rotational frequency (0.375 Hz), in according to study [9] has permitted to obtain a centrifugal force that it was in the range which didn’t cause harmful effects on cells and a flow rate (~500 nl/min) which involved low shear stress inside the cell culture chamber. The flow rate achieved at 0.375 Hz permits to cultivate bacterial cells for about 5 days without change culture medium. The system was used for culturing Pseudomonas aeruginosa at 30˚C and at a flow rate of 400 nl/min. The bacterial culture was observed for two days using a confocal microscope. From the results obtained the platform needs an additional optimization in order to have a better bacterial cells inoculation. After this optimization, the biofilm and the effects of antibiotics will study.