Numerical design of a card and related physicochemical phenomena occurring inside agarose-immobilized bacteria: A valuable tool for increasing our knowledge of biosensors

The detection of heavy metals in the environment constitutes a very important challenge for European policies that focus on health and safety. Biosensors complement the physicochemical methods for the detection of heavy metals and toxic organic substances and yet are also a strong alternative. Several strains of bioluminescent bacteria were created in our laboratory to detect heavy metals, such as the Escherichia coli pBzntlux strain for cadmium detection. These bacteria were immobilized on agarose membrane of a 64-well card which was designed without specific knowledge of fluidic theory. The response of the E. coli pBzntlux strain immobilized in agarose was heterogeneous and necessitated a long induction time. We decided to use the multiphysics software COMSOL, which is capable of combining several differential equations (hydrodynamics, heat and mass transfer) in order to optimize hydrodynamics and transfer phenomena when bioluminescence occurs. As a result, the new card gave an excellent response with a shorter induction time and better homogeneity. To explain the signal of the new improved card, a diffusion study of Cd2+ and oxygen for each card was carried out. The diffusion coefficients of Cd2+ and oxygen in the agarose membrane were assessed to model the transfer kinetics within the immobilized matrix. Simulations showed that after 30 min (induction time) the inducer diffuses through the whole thickness of the well and does not restrict the bioluminescence. However, the oxygen concentration decreases from 100% at the top of the well to 10% at a depth of 3 mm; this result agrees with the experimental profile of bioluminescence (9 × 109 RLU at 1 mm falling to 4.2 × 103 RLU at 3 mm). We can suppose that the bioluminescence was restricted by the oxygen at the bottom of the wells. It justifies the improved signal of the new card (Card 2) as its low thickness resulted in the emitted light being less faded and diluted. esearch provided a new insight into the behavior of chemicals within an immobilized matrix and the numerical design approach led to a better understanding of the hydrodynamics.