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Single-cell analysis has emerged as a powerful tool to elucidate life science questions regarding cellular heterogeneity. In the context of single-cell proteomics, microfluidics forms a promising route to prevent sample dilution and losses during sample preparation. An important aspect of single-cell proteomics is the single-cell isolation strategy. In continuous microfluidics, a common strategy is to use a hydrodynamic trapping array of stiff microstructures to capture individual cells. In the literature, different hydrodynamic trap structures and trapping array designs have been described to capture cells or beads. However, no comparative study has been performed on the effect of these different trap and array designs on the single-cell isolation efficiency. In addition, different operational parameters such as cell concentration, flow rate and the time cells are loaded inside the trapping chamber have never been explored for the different designs. Therefore, the aim of this thesis was to study the effect of different designs and operational parameters on the single-cell isolation efficiency and to optimize this set of parameters for capturing JURKAT cells. In the first step, the JURKAT cell size distribution was evaluated to make necessary adjustments to the trap designs found in the literature. Next, through a screening experiment, it was shown that the trap geometry, as well as the lateral displacement of the traps, have a significant effect on the single-cell trapping efficiency. To further explore the effect of these parameters as well as the effect of the operational parameters, a surface response experiment was performed. With the results of this experiment, a prediction model was fitted that was able to describe the experimental data well. In the next step, the predictive model was used to find parameter conditions to optimize the single-cell isolation efficiency. Finally, the model's accuracy was assessed through a validation experiment, where two conditions were tested. On the one hand, the predictions for a less desired condition were very satisfactory when compared to the actual output. Unfortunately, for the optimal condition, the experimental data did not fall within the confidence interval of the prediction. These results show that the model does not reflect the real situation for the optimal condition well. Therefore, further experiments should be carried out to assess the accuracy of the model for various conditions and to define whether these results are related to the use of different JURKAT cell populations or to the model itself.

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Red blood cells, also called erythrocytes, play a crucial role in the human body by transporting oxygen to various tissues and organs and returning carbon dioxide to the lungs for excretion. Their unique biconcave shape, similar to a doughnut, is maintained by a complex cytoskeleton of proteins and molecules. In this thesis, the synthesis conditions for a building block for an artificial red blood cell cytoskeleton were optimized for use at a later stage in the cytoskeleton of synthetic red blood cells. These building blocks were created using DNA origami, a technique in which a single long DNA strand, known as a scaffold, is folded and structured using shorter DNA strands, called staples, to achieve the desired shape. The examined shape is an asterisk structure, which is designed to mimic the natural structure of the building blocks of the cytoskeleton of red blood cells. In addition, two more hexagonal structures were developed through computer simulations, expanding the range of possible designs. The optimization process focused on several parameters that influence the annealing step of DNA origami synthesis, where the scaffold and staples are assembled and bonded together using a temperature regime. By making variations to the parameters of this process, such as the temperature regime, salt concentration and scaffold-to-staple ratio, a protocol was created that resulted in a structure that resembled the intended asterisk shape. However, during the attempts to purify the obtained structures and remove excess staples, the structures were damaged in the three purification techniques tested. This indicates that further optimization of purification methods is necessary. The results of this thesis are an advance towards the development of synthetic red blood cells. This may have applications in clinical practice, in blood transfusions, so that there is no or less need for traditional blood donations. Furthermore, these synthetic erythrocytes can also be used for research into various aspects of blood cell biology and medical technology.