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 acting during the adsorption: it was found that hydrophobic forces helped by electrostatic ones occur during IgG adsorption onto titania phases. The "stop-flow" method was employed to coat the TiO2 modified microchannels with the capture antibodies and improve the result of a microimmunoassay. When strong adsorbing phases are used, the limit of detection of the assay was lowered by one order of magnitude. The FEM model was used to obtain the kinetic rates of adsorption and desorption of IgG antibodies on PET with a novel biosensor conceived in this lab, which measures capacitive changes in the surface microchannel while the adsorption takes place. As the FEM model foresees a three-dimension region where the adsorption might take place, it can be used to study the adsorption in TiO2 gels developed in our lab.

Dynamic protein adsorption in microchannels by "stop-flow" and continuous flow

Hubert Girault, Andrea Lionello

This work addresses two ways of loading proteins on microchannel surfaces for immunoassay applications: 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. In the present paper, 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”.

2005

Porosity in Aluminum Alloys

Milan Felberbaum

Porosity is one of the major defects in castings because it reduces the mechanical properties of a cast piece [1]. Porosity formation results from the effect of two concomitant mechanisms, namely solidification shrinkage and segregation/precipitation of gases [1]. A model for the prediction of microporosity, macroporosity and pipe shrinkage during the solidification of alloys has been developed at the Computational Materials Laboratory (LSMX-EPFL) [2]. This model has then been improved by taking into account the effect of various alloying elements and gases on porosity formation [3, 4, 5]. However, the modeling of two physical phenomena still needed to be improved: (i) the curvature influence and (ii) the hydrogen diffusion influence on the growth of pores. The effect of pinching, i.e. the pores are forced by the growing solid network to adopt a complex non spherical shape, induces curvature restriction to the pores. This pinching effect can be a limiting factor for the growth of pores and is too simply modeled in the model of Péquet et al. [2]. Several other pinching models exist, but a rigorous experimental study to validate either one of these models is needed. Additionally, Carlson et al. [6] have recently shown that hydrogen diffusion might also be a limiting factor for the growth of pores. In the model of Péquet et al. [2], this effect was not taken into account. This thesis is mainly aimed to (i) provide experimental results that specifically validate the pinching model developed by Couturier et al. [4], (ii) investigate the influence of hydrogen diffusion on the growth of pores and (iii) provide a new model that takes into account the pinching effect and the hydrogen diffusion influence on the growth of pores. At first, pores formed in aluminum-copper (Al-Cu) samples (cast under controlled conditions) have been analyzed using high resolution X-ray tomography. The influence of the alloy inoculant, copper content, cooling rate and initial hydrogen content on the morphology of pores has been investigated. The results show that the curvature of micropores pinched in either non-inoculated or inoculated Al-4.5wt%Cu alloys can be fairly well approximated to that of cylinders. The results also show that the pinching model must be function of (i) the volume fraction of the primary phase gα and (ii) the secondary dendrite arm spacing λ2. However, the influence of the initial hydrogen content appears to be negligible. The pinching model developed by Couturier et al. [4] accounts for these observations and their relation fits fairly well the average mean curvature value of our experimental data. A new model has been developed to calculate an effective hydrogen diffusion coefficient De(gs), that is a function of the volume fraction of solid only. For that purpose, in-situ X-ray tomography has been performed on Al-Cu alloys. For each volume fraction of solid 0.6 ≤ gs ≤ 0.9, a representative volume element of the microstructure has been obtained from the tomography data. Solid and liquid voxels being assimilated to solid and liquid nodes respectively, a hydrogen diffusion equation has then been solved numerically. Calculations have been run until steady-state was reached in order to deduce De(gs) and the simulation results were successfully compared with a new theory based on effective-medium approximations. Both approaches lead to the main conclusion that hydrogen diffusion through the solid phase cannot be neglected, unlike it is assumed in the model of Carlson et al. [6]. Next, using the pinching model of Couturier et al. [4] and the obtained De(gs), a new volume-averaged model has been developed in order to simulate the growth of pores limited by (i) the curvature of the pore phase and (ii) the diffusion of hydrogen. The results show that, although hydrogen diffusion can be a limiting factor for the growth of pores, the pinching effect has a much larger influence. Accordingly, any model for porosity prediction should carefully take into account the influence of curvature and hydrogen diffusion on the growth of pores. In order to ripen this study at a refined scale, a 2D phase-field model has been developed to describe the complex shape of a pore formed within interdendritic liquid channels [7]. The influence of the solid, which can force the pore to adopt a non-spherical shape, is taken into account through the geometry of the domain and appropriate boundary conditions. This model accounts for curvature influence and hydrogen diffusion in the liquid, two of the main aspects governing the growth kinetics of a pore. However, the model still needs to be combined with a description of the liquid flow induced by the pore growth. Basically, this model should serve as a sound basis for further developments that might lead to more sophisticated pinching models. Finally, an experimental study has been conducted in order to track the inoculant influence on the shape of pipe shrinkage. Simultaneously, pipe shrinkage calculations (using the model of Péquet et al. [2]) were performed in order to track the influence of the gs,c parameter on the shape of the pipe shrinkage. This gs,c parameter corresponds to the critical volume fraction of solid at which mass feeding stops. Comparisons between experimental and simulation results show that the gs,c parameter should be set equal to 0.6 or 0.1 for a casting simulation of an inoculated or non-inoculated alloy, respectively.

EPFL2010

Porosity in Aluminum Alloys

Milan Felberbaum

EPFL2010