Bubble-Free Operation of a Microfluidic Free-Flow Electrophoresis Chip with Integrated Pt Electrodes

Abstract

In order to ensure a stable and efficient separation in microfluidic free-flow electrophoresis (FFE) devices, various methods and chips have been presented until now. A major concern hereby is the generation of gas bubbles caused by electrolysis and the resulting disturbances in the position of the separated analyte lanes. Instable lane positions would lead to a decreased resolution in sample collection over time which certainly would be problematic when incorporating a stationary detector system. In contrast to our previous publications, in which we implemented laborious semipermeable membranes to keep bubbles outside the separation region, here we describe an electrochemical approach to suppress the electrolysis of water molecules and therefore bubble formation. This approach allowed a simpler and additionally a closed chip device with integrated platinum electrodes. With the use of this chip, the successful separation of three fluorescent compounds was demonstrated. Quinhydrone, which is a complex of hydroquinone and p-benzoquinone, was added only to the local flow streams along the electrodes, preventing mixing with the separation media and sample. The electrical current was generated via the oxidization and reduction of hydroquinone and p-benzoquinone up to a certain limit of the electrical current without gas formation. The separation stability was investigated for the chip with and without quinhydrone, and the results clearly indicated the improvement. In contrast to the device operating without quinhydrone, a 2.5-fold increase in resolution was achieved. Furthermore, separation was demonstrated within tens of milliseconds. This chemical approach with its high miniaturization possibilities offers an interesting alternative, in particular for low-current miniaturized FFE systems, in which large and open electrode reservoirs are not tolerable.

Reassemblable quasi‐chip free‐flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

“…The two electrodes were located near the bottom of electrode bath. The bubbles within the two electrode baths could be quickly removed via a pump, effectively avoiding the interference of bubbles to IEF run [43].…”

Mathematical and numerical model to study two-dimensional free flow isoelectric focusing

“…Figure 3(a) (symbols) shows an almost linear pH profile at the end of their microchannel, where the pH values were obtained from the locations of 7 pI markers used in their experimental study. 5,6 To validate our model, we have simulated an identical case considering the 7 pI markers used in the experimental work of Kohlheyer et al 5 The physico-chemical properties of pI markers are listed in Table II. The pH gradient is created using 48 biprotic (DpK ¼ 2.5) ampholytes 24 having isoelectric points between pH of 3.7 and 10.3.…”

“…4 However, even though their work has demonstrated the feasibility of FFIEF in a microchip, it was severely restricted to low applied electric fields because of bubble formation at the electrode surfaces. To circumvent the bubble problem, Kohlheyer et al 5,6 introduced a modified FFIEF micro-device by adopting anodic and cathodic sheath flows. In their experimental work, a linear pH gradient was developed and the separation of pI makers was accomplished.…”

Recent developments in electrophoretic separations on microfluidic devices

“…In both examples, the channel was on a microchip; however, the channel length was 5 cm and the width was 3 cm. Kohlheyer et al described a new method for preventing electrolysis in a microfluidic free-flow device [23]. Quinhydrone (QH), a complex of hydroquinone (H 2 Q) and p-benzoquinone (Q), was added to the system as strategy to electrochemically quench hydrolysis.…”