title: Investigations toward the accumulation, separation, mixing and detection of charged species using microfluidic electrophoresis
Creators: name: Gebhard, Florian
Publisher: name: UNSPECIFIED
Year: 2024

description: In microfluidics, a variety of electrophoretic techniques is used, for example to purify, focus, fractionate, detect or process bioparticles such as DNA molecules, proteins, cells or extracellular vesicles. Although there are many established methods - e.g. capillary electrophoresis, free-flow electrophoresis or isotachophoresis - there is still much potential
for improvement and development of new approaches. Therefore, this thesis presents investigations toward the accumulation, separation, mixing and detection of charged species
using microfluidic electrophoresis.

The first part deals with the electrophoretic transport of bioparticles in aqueous twophase systems (ATPS) based on dextran and polyethylene glycol (PEG). In a first step, the interaction of two model proteins with the ATPS interface is investigated. Both proteins experience an interfacial transport resistance when they are transported from the dextran-rich phase to the PEG-rich phase. However, as this transport resistance is not equally
pronounced for both proteins, they can be separated at the phase boundary. A similar principle is then applied to a solution containing both exosomes and proteins. While the exosomes are retained at the phase boundary, the proteins can cross over relatively easily. It should therefore be possible to purify exosomes in this way.

Next, the stability of the liquid-liquid phase boundary in an ATPS is studied. Especially at higher field strengths, instabilities occur that are presumably caused by Faradaic reactions at the electrodes. Only if these phenomena are prevented, it will be possible to study the actual influence of the electric field on the interface and to reliably perform separation processes.

The next part of this thesis focuses on sample detection using isotachophoresis (ITP), a special electrophoretic technique that allows to increase the local concentration of a sample by several orders of magnitude. A new approach is presented to lower the detection limit via signal processing by exploiting knowledge of the physics of electrophoretic sample transport and the imaging process. By cross-correlating pairs of noisy fluorescence images of an analyte focused by ITP, the electrophoretic velocity of the sample can be extracted in a first step even at low signal-to-noise ratios. Based on this velocity, a Galilean transformation is then performed on the entire set of images to align the fluorescence intensity distributions of the sample obtained from the individual images and generate a series of quasi-replicate measurements. Averaging over the transformed data significantly reduces the noise superposing the raw images. In this way, the detection limit is lowered by approximately two orders of magnitude without any additional instrumentation.

Microfluidic ITP offers advantages not only in detecting low-concentration samples but also in significantly accelerating the reaction rate of chemical species by co-focusing reactants within a narrow sample zone. However, traditional ITP lacks the capability to control the reaction rate in real time. Therefore, a novel ITP mode is introduced that enables the temporal manipulation of the overlap of two ITP zones by applying an oscillating electric field. By adjusting the frequency and amplitude of the oscillation, it is possible to precisely control the time average of this overlap. This concept is demonstrated using two non-reactive fluorescent species. However, it is proposed that this approach can be applied to chemical reactions between ionic species focused by ITP, allowing for the direct control of the corresponding reaction rate by tuning the parameters of the oscillatory electric field.