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

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

Chemical and physical processes for integrated temperature control in microfluidic devices

Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature. In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool a microchannel. Additionally, heating of a microchannel was achieved by dissolution of concentrated sulfuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allows control of the position and the magnitude of the thermal effect

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 μM level were obtained in combination with good peak shapes and an overall good performance and stability

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

On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-μm-wide, and 20-μm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 μM for potassium was demonstrated

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 μm × 70 μm 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 μM up to 2 mM. Detection limits were at the low μm level. Separation of two short peptides with a pl 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