Controlled protein adsorption for microimmunoassays

This dissertation addresses the adsorption of proteins in microchannels with the aim of improving the result of an immunoassay. To observe the adsorption of fluorescently labelled immunoglobulins G in laser ablated polymer microchannels, a confocal microscope was built. By optically scanning the specimen, and observing one focal plane at time, this tool allows for a high signal-to-background, which leads to a low limit of detection (10-9 M). A numerical model for the adsorption kinetics of proteins on the walls of a microchannel has been developed using the finite element method (FEM). The model illustrates the adsorption limitation sometimes observed when the microdimensions of these systems induce a global depletion of the bulk solution. A new non-dimensional parameter is introduced to predict the final value of the coverage of any microsystem under static adsorption. A working curve and a criteria (h/KΓmax > 10) are provided in order to choose, for given adsorption characteristics, the value of the volume-to-surface ratio (i.e. the channel height h) avoiding depletion effects on the coverage (relative coverage at least 90% of the theoretical one). Simulations were compared with confocal microscopy measurements of IgG antibody adsorption on the walls of a PET (poly(ethylene terephthalate)) microchannel. The fit of the model to the experimental data show that the adsorption is under apparent kinetic control. Two ways of loading proteins on microchannel surfaces for immunological applications have been analysed with the FEM model: the "stop-flow" and the continuous flow processes. The "stop-flow" method consists of successive static incubation periods where the bulk solution depletes upon the adsorption process. A multi-step "stop-flow" protein coating is studied and compared to a coating under continuous flow conditions. For the "stop-flow", a non-dimensional parameter is here introduced, indicating the adsorbing capacity of the system, by which it is possible to calculate the number of loads necessary to reach the optimum coverage. For the continuous flow, the effects on the adsorption of the kinetic rates, flow velocity and wall capacity have been considered. This study shows the importance of a careful choice of the fluid velocity to minimise the sample waste. For diffusion controlled and kinetics controlled processes, two flow velocity criteria are provided in order to obtain the best possible coverage, with the same amount of sample as with the "stop-flow". Surface modifications have been conceived in order to improve the adsorption and the activity of the physisorbed antibodies. The microchannels have been coated with titania nanomaterials, chosen because of their biocompatible properties. Crystalline and amorphous TiO2 have been used and the adsorption with respect to the native PET was improved by 3 times. A study of adsorption as a function of the pH solution and different ionic strengths has been done in order to infer the forces actin