Erik Baltussen

Recent advances in affinity capillary electrophoresis

Use of the specificity of (bio)interactions can effectively overcome the selectivity limitation faced in capillary electrophoresis (CE), and the resulting technique usually is referred to as affinity capillary electrophoresis (ACE). Despite the high selectivity of ACE, several important problems still need to be addressed. A major issue in all CE separations, including ACE, is the concentration detection limit. Using UV detection, this is usually in the order of 10—6 M whereas laser‐induced fluorescence (LIF) detection can provide detection limits down to the sub‐10—10 M range. However, a marked disadvantage of LIF is that labeling of the analytes is usually required, which might change the interaction behavior of the solutes under investigation. Additionally, labeling reactions at sub‐10—10 M concentration levels are certainly not trivial and often difficult to perform quantitatively. Alternative and universal detection approaches, particularly mass spectrometric (MS) detection, look very promising but (A) CE‐MS techniques are still far from routine application. Important future progress in sensitive detection strategies is likely to increase the use of ACE in the future.

New approaches for fabrication of microfluidic capillary electrophoresis devices with on-chip conductivity detection.

In practice, microfluidic systems are based on the principles of capillary electrophoresis (CE), for a large part due to the simplicity of electroosmotic pumping. In this contribution, a universal conductivity detector is presented that allows detection of charged species down to the μM level. Additionally, powderblasting is presented as a novel technique for direct etching of microfluidic networks. This method allows creation of features down to 50 μm with a total processing time (design to device) of less than one day. The performance of powderblasted devices with integrated conductivity detection is illustrated by the separation of lithium, sodium, and potassium ions and that of fumaric, malic, and citric acid.

Capillary electrophoresis with on‐chip four‐electrode capacitively coupled conductivity detection for application in bioanalysis

Microchip capillary electrophoresis (CE) with integrated four‐electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip‐based analysis, is compatible with miniaturization and on‐chip integration. The glass microchip structure consists of a 6 cm etched channel (20 νm×70 νm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 νM up to 2 mM. Detection limits were at the low νM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.

Considerations on contactless conductivity detection in capillary electrophoresis

Nearly all analyses by capillary electrophoresis (CE) are performed using optical detection, utilizing either absorbance or (laser‐induced) fluorescence. Though adequate for many analytical problems, in a large number of cases, e.g., involving non‐UV‐absorbing compounds, these optical detection methods fall short. Indirect optical detection can then still provide an acceptable means of detection, however, with a strongly reduced sensitivity. During the past few years, contactless conductivity detection (CCD) has been presented as a valuable extension to optical detection techniques. It has been demonstrated that with CCD detection limits comparable, or even superior, to (indirect) optical detection can be obtained. Additionally, construction of the CCD around the CE capillary is straightforward and robust operation is easily obtained. Unfortunately, in the literature a large variety of designs and operating conditions for CCD were described. In this contribution, several important parameters of CCD are identified and their influence on, e.g., detectability and peak shape is described. An optimized setup based on a well‐defined detection cell with three detection electrodes is presented. Additionally, simple and commercially available read‐out electronics are described. The performance of the CCD‐CE system was demonstrated for the analysis of peptides. Detection limits at the νM level were obtained in combination with good peak shapes and an overall good performance and stability.

Use of bioaffinity interactions in electrokinetically controlled assays on microfabricated devices.

In this contribution, the role of bioaffinity interactions on electrokinetically controlled microfabricated devices is reviewed. Interesting applications reported in the literature include enzymatic assays, where enzyme and enzyme inhibition kinetics were studied, often in combination with electrophoretic separation. Attention is paid towards developments that could lead to implementation of electrokinetically controlled microdevices in high‐throughput screening. Furthermore, enzyme‐facilitated detection in combination with electrophoretic separation on microdevices is discussed. Various types of immunoassays have been implemented on the microchip format. The selectivity of antibody‐antigen interaction has been exploited for the detection of analytes in complex sample matrices as required, for example, in clinical chemistry. Binding kinetics as well as stoichiometry were studied in chip‐based assays. Automated mixing protocols as well as the demonstration of a parallel immunoassay allow implementation of microdevices in high‐throughput screening. Furthermore, demonstration of immunoassays on cheap polymeric microdevices opens the way towards the fabrication of disposable devices, a requirement for commercialization and therefore for application in routine analyses.

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 νm Hypersil ODS particles, separation efficiencies in the range of 130 000–200 000 plates/m were obtained. In a 100 νm inner diameter packed column, electrical currents up to 50 νA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in‐ and outlet vial during separation was not required to prevent bubble formation.

Indirect electro-osmotic pumping

The manipulation of liquids within a microcapillary network remains a considerable challenge in the development of miniaturized total chemical analysis systems (μTAS). Fluid manipulation can be achieved using (micro) mechanical pumps connected or integrated into the device, and by using an electric field (E) for generation of electro-osmotic flow (EOF). For glass microdevices, electro-osmotic pumping (EOP) is most attractive, since no moving parts and/or valves are required. In its simplest embodiment, EOP in microfluidic devices involves imposing an E along the full length of the channel by immersing electrodes into open solution reservoirs situated at both ends of the channel. Electrolytically generated gases at the electrodes drift to the surface of the solution reservoirs and escape into the air. In more complex situations, however, EOP in a subsection of a microchannel may be required. For sampling, for example, from brain tissue in living organisms, the presence of electrodes in the ‘sample reservoir’ (i.e., the brain), and thus outside the microdevice is undesirable, since potentials applied to external electrodes interfere with the sampling environment. In these cases, electrodes need to be integrated into the microfluidic device. The use of electrodes in a microchannel, however, is not trivial. Electrolytic gases get caught in the sealed microchannel and hence effectively interrupt the electric field, and thus fluid movement. A number of approaches to avoid bubble formation during spatially localized application of voltages in microfluidic networks have been reported. In one example, a 1-mm-thick poly(dimethylsiloxane) (PDMS) substrate containing the microchannel was sealed with a glass cover plate containing the electrodes.1 Electrolytic gases formed at the electrodes dissipated through the highly gas-permeable PDMS film into the air. An alternative method for application of the electric field is the use of a conducting barrier between the electrodes and the channel. A Nafion membrane has been presented as an interface between an open reservoir containing the electrode and a microchannel.2 Electrolytic gases dissipate into the air via the open reservoir, while the electrical contact afforded by the membrane ensured that an E was applied to the closed microchannel. A similar approach involves the use of adjacent side channels, which are electrically connected, via porous barriers, but where fluid exchange is strongly limited.3,4 Either the porous membrane was formed using a thin layer of potassium silicate, in or the contact was directly over the glass wall separating adjacent channels. The three approaches mentioned above allow the creation of field-free zones in addition to regions where the field is applied. In the field-free regions, charge-independent fluid transport can be controlled by EOP elsewhere in the microfluidic system, an effect we term “electro-osmotic indirect pumping” (EOIP) to distinguish between EOP in- and outside the electric field. In this paper, a glass microdevice for both EOP and EOIP using electrically connected side channels is presented. Electrical contact between the main and side channels is achieved by electrical breakdown of the glass barrier between these channels. Electrical breakdown for initiating liquid contact between disconnected channels has been demonstrated in PDMS devices.5 To our knowledge, this is the first time that electrical breakdown for initiation of electrical contact between glass microchannels is presented. Cross injection by a combination of EOP and EOIP is demonstrated.

Indirect Electro-Osmotic Pumping for Direct Sampling from Bioreactors

In this contribution, electro-osmotically induced, pressure-driven flow along a micromachined channel is presented, using two adjacent channels at either end. Imperfect bonding in the narrow glass wall between the main channel and each side channel allows electrical contact. A voltage applied between the side channels results in an electric field in the main channel, thereby enabling capillary electrophoresis (CE) and generating an electro-osmotic flow (EOF). Electrical displacement of liquid in one section of the channel induces hydraulic fluid flow in the other, field-free sections of the channel, a phenomenon we call indirect electro-osmotic pumping (IEOP).