Digital Microfluidic Sampler for a Portable Capillary Electropherograph

Liquid-liquid extraction techniques are one of the major tools in chemical engineering, analytical chemistry, and biology, especially in a system where two immiscible liquids have an interface solutes exchange between the two liquid phases along the interface up to a point where the concentration ratios in the two liquids reach their equilibrium values [1]. Solutes including nucleic acids and proteins of interests can be extracted from one liquid phase to the other immiscible liquid phase as a preparation step for many analytical processes. There are several advantages in miniaturizing the liquid-liquid extraction methods to on-chip level extraction. Usual advantages of miniaturization are the reduction in the sample size and portability. In addition, transport phenomena is faster in Micro-systems than in ordinary size systems, and therefore, one may expect that liquid-liquid extraction takes less time to achieve in miniaturized devices. It is due to shorter diffusion time in micro scale as well as high surface to volume ratio of Microsystems. Electrowetting on dielectric (EWOD) digital microfluidics is an efficient platform to process droplet based analytical processes [2]. Nanoliter (nL) or smaller volume of aqueous liquid droplets can be generated and transported on a chip by EWOD process. In addition to the high surface to volume ratio, high chemical potential can be expected in droplet based extraction when the droplets are in motion. In this paper, we propose to use room temperature ionic liquid (RTIL) as a second liquid phase for extraction, which forms immiscible interface with aqueous solutions. Properties of RTIL can be tailored by choice of cation, anion and substituents. RTIL has been investigated as replacements for the organic solvents and various “task-specific” ionic liquid are being developed which exhibit many attractive properties such as very low vapor pressure, high thermal stability [3]. We recently published EWOD properties of various RTILs toward microfluidic applications [4]. To demonstrate liquid-liquid micro extraction on chip, we fabricated and tested EWOD digital microfluidic devices. Fig. 1 shows (a) top and (b) cross sectional views of EWOD device. Two model extraction systems were tested. One is organic dye extracted from RTIL (1-butyl-3-methylimidazolium bis(trifluoromethanesulfonylimide or BMIMNTf2) to water and the other is iodine (I2) extracted from water to BMIMNTf2. The later model experiment is demonstrated in Fig. 2. Droplets of aqueous solution and BMIMNTf2 solution were generated on chip reservoir then transported for extraction and separated by EWOD actuation. When an aqueous solution and BMIMNTf2 solution join together, they created an interface, since water and BMIMNTf2 are immiscible. Extraction of I2 was done along the interface. After successful extraction, two immiscible liquid phases were separated by EWOD actuation and formed two separate droplets. From the result shown in Fig 2 (g), it is expected that extraction performance at the interface of moving droplet would be enhanced compared to the stationary droplet, because a moving interface prevent the chemical equilibrium, thus more chemical extraction potential can be provided with a moving interface than at a stationary interface. This demonstration is the first step toward total analysis system. The presented result opens the way to on-chip micro extraction, which will be readily integrated with other sample preparation microfluidic components and detection components. Currently, micro extraction systems for larger molecules such as nucleic acids, proteins and biological cells are being developed for further analytical applications.

To meet the increasing demand of efficient cooling performance in small scale, this paper presents a digital microfluidics (DMF) microscale liquid cooling system which works on the principle of electrowetting on dielectric (EWOD). In EWOD DMF, fluids are handled drop-wise by external electric field. When the dispensed liquid droplet arrives at the hotspot by EWOD DMF operation, it picks up heat and removes heat when it leaves. This process can be repeated for a series of droplets by using a completely automated LabVIEW controlled system connected to the PCB package. With the help of indium tin oxide (ITO) thin film resistance temperature detectors (RTD) and pre-calibrated temperature coefficient of resistance (TCR) data, the temperatures of the hotspot before and after the residence of liquid droplet (i.e., cooling) can be recorded for different frequencies (dwelling time period of droplet on the hotspot) of the drop motion and varying heater power. Future work will involve RTD resistance data collection to plot the heat flux and the temperature difference (before and after cooling) for different frequencies of drop motion. Although the primary focus is to study single phase cooling, the DI water drop will experience considerable evaporation resulting in higher cooling performance. The single phase cooling studies will help in establishing a robust platform for future two-phase cooling analysis in which evaporation effects will be considered.

This thesis focuses on the optimisation of capacitively coupled contactless conductivity detection for capillary and microchip electrophoresis and its applications in analytical chemistry. First, the effect of high excitation voltages and operation frequencies on the capacitively coupled contactless conductivity detector cell for conventional capillary electrophoresis is evaluated. The detector electrodes comprised two steel tubes cut from hypodermic needles, through which the capillaries were inserted. It is demonstrated that increasing excitation voltages from 25 V pp, to 250 Vpp improves the detection limits by a factor of 10. The high actuator voltage approach was also investigated for contactless conductivity detection on a glass-microchip device with ancm long channel. The detector electrodes formed part of the microchip and were placed on the microchip directly above the microchannel. In a separate project the simplification of on-microchip contactless conductivity detection was accomplished. This was achieved by integrating the detector electrodes on to a chip-holder specifically designed for this purpose. Thus the electrodes were a part of the holder, an improvement of the previous arrangement whereby the detector electrodes were situated on the microdevice. Finally the applications and advantages of the optimised high-voltage capacitively coupled contactless conductivity detection for inorganic and organic analysis were demonstrated. The separation and detection of 14 metal ions was accomplished in less than six minutes. The compatibility of this detector with non-UV transparent, polymer capillaries has been demonstrated. The detection of native amino acids has been evaluated. Part of the work was dedicated to the on-chip analysis of various classes of organic ions. The two immunoproteins human immunoglobulin M (IgM) and immunoglobulin G (IgG), were analysed in their unlabelled state on both capillary and lab-on-chip platforms. All species involved in an immunological interaction between IgM and IgG could be detected. A method for the analysis of selected basic pharmaceutical drug substances was developed. Detection limits comparable to those supplied by direct UV detection were obtained. Main component assays of selected pharmaceutical preparations have been demonstrated.

By transforming the time-based x-axis of electropherograms in capillary zone electrophoresis (CZE) into the corresponding effective mobility-scale, we propose a simple and robust data representation for a better qualitative and quantitative capillary electrophoresis (CE) analysis. The time scale of the raw electrophoretic data (detection signal versus time) is transformed into an effective electrophoretic mobility scale (mu eff-scale) with account of the electroosmotic flow (EOF) peak or of an internal standard of known effective mobility. With the new scaling (detection signals versus effective mobility), the obtained electropherograms are more representative of the velocity-based electrophoretic separation and the comparison of complete electropherograms is directly possible. This is of importance when tracking peaks in real samples where alteration in EOF stability can occur or when comparing electrophoretic runs from different experimental setups (independence in column length and voltage). Beside the qualitative possibilities, a quantitative improvement is achieved in the mu eff-scale with significant better peak area reproducibility and equal to more precision in quantitative analysis than with the primary time-scale integration.

A miniaturized capillary electrophoresis (CE) system with contactless conductivity detection (CCD) and flow injection (FI) sample introduction was developed. It represents an ideal combination of high‐resolution separation of CE with high throughput reproducible sample injection of FI, and universal and robust detection of CCD for the analysis of ionic species. Its design and fabrication method is described in detail. Performance tests of the system were performed for the analysis of surface water. It was found that complete separation of the cations studied (NH4 +, K+, Ca2+, Na+, and Mg2+) could be obtained within 60 sec with peak height RSD better than 2.0%, thus achieving a sampling frequency of 60 hr−1. Detection limits were in the order of low micro molarity.

Simultaneous determination of creatinine and acetate by capillary electrophoresis with contactless conductivity detector as a feasible approach for urinary tract infection diagnosis

Wet deposition and related atmospheric chemistry in the São Paulo metropolis, Brazil: Part 1. Major inorganic ions in rainwater as evaluated by capillary electrophoresis with contactless conductivity detection

This paper describes highly efficient in-droplet particle concentrations/separations (over 91% in efficiency), where magnetic particles are concentrated/separated within a droplet by applying a magnetic field and a subsequent EWOD (electro wetting-on-dielectric) actuation physically splits a magnetic particle-rich region into an individual droplet. This in-droplet method will provide a new functionality of separation/concentration for digital (droplet-based) microfluidics, possibly allowing use of many magnetic-particles-based bio assays in digital microfluidics.

The analyses of nucleotides are important for a variety of biological samples, including blood, plasma, tissue and DNA and RNA hydrosylates. High performance liquid chromatography (HPLC) has been used in the past for nucleotide separations but is limited by the microliter volume sample requirement, resolution and the speed and high cost of the analyses. Capillary electrophoresis (CE) is a useful alternative to HPLC for the analysis of nucleotides. CE separations of nucleotides have been reported for both the CZE and MECC modes. Several different types of buffers, such as phosphate, borate and organic buffers, including CAPS have successfully been used to separate nucleotides. MECC separations have used SDS to provide a partitioning phase within the buffer for the simultaneous separation of nucleosides and nucleotides. Researchers have used both methods to separate nucleotides quickly and with plate counts above 100,000 plates/m. However, while CE separations are faster, more selective and less expensive than comparable HPLC methods, the mechanism of separation in CE analyses is not fully understood, thus method optimization is performed on a trial and error basis. In addition, the nanoliter size sample used in many CE analyses limits the sensitivity of most detection methods, including UV, fluorescence, and MS. In the present work, CZE methods for nucleotides, nucleosides and bases were optimized using bare capillaries and sodium bicarbonate/carbonate buffer systems. The parameters of concentration, voltage and pH were investigated to determine their effects on the separations. The data collected from these optimizations was used to correlate the experimental mobilities of the analytes to their ionic charge to mass ratios. A linear relationship would permit the prediction of the migration order of the analytes before analysis, thus minimizing the time and expense of method optimization. In addition to method optimization, the sample preconcentration method of whole capillary stacking was applied to nucleotides to lower the detection limits of the CE-UV technique from the micromolar range to nanomolar quantities. After optimization of both the stacking and separation methods with ribonucleotides, the technique was applied to a separation of carbovir triphosphate, a nucleoside analog with anti-HIV properties, from dATP and dGTP.

Analysis reproducibility and detection sensitivity of capillary electrophoresis (CE) are often questioned by applied scientists, which has hindered its application as a routine method. To address these issues, a simple, precise, and reproducible dynamic coating method was developed by applying carboxymethyl chitosan (CMC) dynamic coating on fused silica capillary. The proposed coating was accomplished by simply rinsing the capillary with CMC solution for 1 min in between runs, with no regeneration procedure or buffer additives needed. Electroosmotic flow could be well controlled by adjusting the pH of background electrolyte, and the adsorption of analytes onto the capillary inner wall was effectively eliminated. The main parameters of the coating condition were optimized, and extensive applications of these CMC-dynamically coated capillaries in CE separations were then firmly confirmed. By using proteins, aristolochic acids, and inorganic anions as model analytes, the coating showed a good stability, high reproducibility, as well as improved sensitivity. Baseline separations could be obtained with high efficiency. The reduced adsorption was impressively effective for basic proteins, with an average plate number of 90,000/m for each protein, apart from the good resolution on the chromatogram. A high sensitive detection of α-lactalbumin was achieved with a limit of detection (S/N = 3) of 3.5 nM, and the number of theoretical plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with nonaqueous CE and CE-mass spectrometry proved to be practical. All results showed that the CMC-dynamically coated capillary has special properties and obvious superiority over the uncoated ones for CE analysis.

Capillary electrophoresis (CE) is an efficient and fast microseparation technique that is well suited for the separation of nucleic acids. CE is routinely used for the separation of double-stranded DNA fragments; single-stranded DNA fragments and polymerase chain reaction (PCR) products in our laboratory. CE is a versatile tool for separating nucleic acids in molecular biology (1). CE has the advantages of automation, small sample requirement, fast and efficient separation, real-time detection, and negligible buffer waste in comparison to slab-gel electrophoresis. However, the major drawback of CE is low amount of sample throughput because samples are analyzed sequentially. This problem is resolved by using a capillary array electrophoresis (CAE) (2,3). Since conventional CE systems are equipped with a single capillary, a practical way to increase throughput is to minimize the analysis time per sample. This has been accomplished by using a range of an effective length capillary as short as 7 cm and field strength as high as 2000 V/cm for the separations of small ions, drugs, and proteins (4–7). Recently, we were able to achieve fast separation of the wild-type and mutant PCR products of the TGF-β1 gene in 60 s using a 7-cm effective length capillary and 560 V/cm (8). However, high field strength degraded the resolution of large DNA fragments (9) and may cause migration anomalies (10). Alternatively, one can use a shorter capillary length with low field strength. We describe here the fast sizing of DNA fragments using 1-, 2-, and 7-cm effective length capillaries (11). Fast DNA separation can be achieved with capillaries with an effective length of only 1–2 cm. The low electric field associated with such short capillaries extends the range of DNA fragment sizes that may be separated by this means, which is useful if the analysis of large DNA fragments is required.

Molecularly imprinted polymers based enrichment and separation for trace analysis in capillary electrophoresis.

This paper reviews the principles, operations, and applications of bubble-based electrowetting-on-dielectric (EWOD). EWOD has proved to be an efficient tool in digital microfluidics that employs discrete droplets, and various applications that use the principles of EWOD have been developed from lab-on-a-chip to optical systems. Similar to its use with droplets, EWOD can also be applied to gaseous bubbles. This review begins with a discussion of the principles of EWOD for a bubble on an electrode covered with a hydrophobic dielectric layer. It then addresses EWOD actuation and the transportation of a bubble in an aqueous medium, along with a physical explanation of bubble motion. The operation of EWOD is then extended to the on-chip creation/elimination and splitting of bubbles. In particular, micro-mixers and pumps are discussed as potential applications of these operations. Unlike droplets, bubbles can be easily oscillated by external excitation, which provides additional functionalities. By integrating EWOD with external excitation, a number of new advanced applications are introduced, including the capture/separation of particles and the propulsion of objects. In these advanced operations, cavitational microstreaming flows and acoustic radiation forces are mainly responsible for the physical mechanisms. This paper also discusses these advanced operations along with their underlying physics. It is expected that in addition to bubble oscillation, other bubble actuation modes will create new functionalities and new potential applications.

The at-line coupling of capillary electrophoresis (CE) and surface-enhanced resonance Raman spectroscopy (SERRS) was optimized for the separation and subsequent spectroscopic identification of charged analytes (dye compounds). Raman spectra were recorded following deposition of the electropherogram onto a moving substrate. To this end a new interface was developed using a stainless steel needle as a (grounded) cathode. The outlet end of the CE capillary was inserted into this metal needle; CE buffer touching the needle tip served as the electrical connection for the CE separation. A translation table was used to move the TLC plate at a constant speed during the deposition. The distance between the tip of the fused silica column and the TLC plate was kept as small as possible in order to establish a constant bridge-flow, while avoiding direct contact. The dyes Basic Red 9 (BR9), Acid Orange 7 (AO7) and Food Yellow 3 (FY3) were used as test compounds. After CE separation in a 20 mM borate buffer at pH 10, after deposition, concentrated silver colloid was added to each analyte spot, followed by irradiation with 514.5 nm light from an argon ion laser to record the SERRS signal using a Raman microscope. Different types of silver colloids were tested: Lee-Meisel type (citrate), borate, and gold-coated silver. BR9 (positively charged) gave much more intense SERRS spectra than the two negatively charged dyes. For BR9 and AO7 the citrate-coated Lee-Meisel colloid yielded the most intense SERRS spectra. The CE-SERRS system was used to separate and detect the negatively charged dyes. Silver colloid and nitric acid (to improve adsorption) were added post-deposition. Even though their chemical structures are very similar, AO7 and FY3 could be readily distinguished based on their SERRS spectra. The limits of detection (S/N = 3) of the CE-SERRS system ranged from 6.7 x 10(-5) M (2.6 x 10(-12) mol injected) for FY3 down to 1.8 x 10(-6) M (7.0 x 10(-14) mol injected) for BR9.

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