Elektrische vangst van biodeeltjes in microfluidica

Since proteins play a major part in cell functioning, there is a large interest in analysing the protein composition of samples to understand which proteins play a significant role, for example within the context of disease progression and pathogenic responses. The samples of which the protein compound has to be determined are becoming progressively smaller and smaller. Mass spectrometry is a powerful technique to identify the sample's proteins, since it reveals both qualitative and quantitative information, but its accuracy depends on the purity of the introduced sample. Hence, prior to identification with mass spectrometry, the sample complexity is reduced by performing a protein separation step. This protein separation step is challenging for small samples, as typically observed problems become more severe once the sample size decreases, such as, e.g., sample losses during separation and the presence of peak broadening by molecular diffusion or by the non-uniform flow profile. These factors deteriorate the purity and decrease the available amount of the analyte that can be introduced to the mass spectrometer. Microfluidic protein separation techniques answer well to these problems. For example, by integration of different steps onto a single platform, the sample losses are reduced; or, the peak broadening by molecular diffusion can be limited due to faster analysis times while the analyte dispersion by the non-uniform flow can be reduced because of the smaller dimensions. Today's most commonly used separation techniques are capillary electrophoresis and high pressure liquid chromatography. When mass spectrometry is used for identification of the proteins in combination with these separation techniques, good results are obtained, since the flow-induced peak broadening is limited for both techniques due to the uniform flow velocity across the channel's cross-section. However, both techniques still suffer from the peak broadening due to molecular diffusion. This can be improved when implementing these techniques in a microfluidic format, but the peak broadening due to molecular diffusion is never completely removed. Electric field gradient focusing (EFGF) is a separation technique which can counteract this peak broadening by molecular diffusion. The technique works by balancing a constant hydrodynamic drag force, depending on the protein's size, with a gradient electrophoretic force depending on the protein's charge, exerted by a DC electric field gradient. This way, each protein has a unique focusing point, and a restoring force is applied once the protein moves away from this focusing point, which effectively counteracts the peak broadening by molecular diffusion. In today's implementations of EFGF, the resolution is still limited due to the presence of flow-induced peak broadening, because a pressure-driven flow is used. In some implementations a packing material is added to the channel, which reduces the flow-induced peak broadening, but can never fully eliminate it. The principle of an electro-osmotic flow (EOF) is known to result in a plug flow in a fluidic channel. Whereas a DC EOF can not be used due to the presence of a DC electric field gradient in the case of EFGF, an AC EOF could be used, as it can in principle be generated independently of the global DC electric field. AC EOF is an EOF generated by electrodes placed along the channel length: upon application of an out-of-phase AC-signal to a pair of electrodes, charges accumulate on these electrodes forming an electric double layer (EDL), while simultaneously a tangential electric field is formed actuating this EDL. In this thesis, we investigated the feasibility of the new concept of using AC EOF in microfluidic EFGF, to remove the flow-induced peak broadening. This could result in a unique separation method in which both the peak broadening by molecular diffusion and the flow-induced peak broadening are removed. The concept of EFGF with AC EOF was first verified by numerical simulations. We found that a constant AC EOF can be generated for different DC electric field strengths present in the electrolyte, if a DC offset was applied to the electrodes to match the local electrolyte DC potential. With a decoupled AC EOF and DC electric field gradient, we observed an improved modelled separation resolution compared to EFGF with a pressure-driven flow or a pressure-driven flow through a packing material. The concept was then studied experimentally. First, it was derived for which electrode and channel geometry, which ion concentration, which electrode material and which AC-signals the AC EOF plug flow could be created. The electrode dimensions were designed based on the system's equivalent electrical circuit, the channel dimensions were chosen to maximize the part of the periphery covered with electrodes, to reduce the influence of friction with the channel walls on the plug flow. It was found that the electrodes, which were gold in this case, were ideally covered with a HfO2 passivation layer, to allow large AC-signals to be applied to the electrolyte while avoiding reactions between the electrode and the electrolyte. An electrolyte with a KCl-concentration of 0.1 mM was found to result in a significant flow velocity of ∼ 100 μm/s for AC-signals with an amplitude of 6 V. Second, it was derived how to suppress the DC EOF, since the presence of a DC EOF in combination with a DC electric field gradient would distort the generated plug flow. It was found that applying a coating to the channel surface or adapting the pH of the electrolyte to match the channel surface's pI could minimize the DC EOF. Last, a device was designed that could apply the DC offsets to the electrodes generating the AC EOF. As it was too cumbersome to generate a DC offset for each electrode off chip due to the large number of electrodes, it was chosen to apply an externally generated DC offset per group of electrodes, and compensate the remaining DC electric potential difference by an integrated capacitor for each electrode. These capacitors were designed based on the system's equivalent electrical circuit. It was derived experimentally that by this design, a plug flow profile with a constant velocity could be obtained while different DC electric field strengths were applied to the electrolyte. Furthermore, it was demonstrated that AC EOF could be used to immobilize model particles in a DC electric field. This indicates that the AC EOF is a promising candidate for plug flow generation in EFGF, and thus to remove the flow-induced peak broadening in this technique.