Recent progress of online sample preconcentration techniques in microchip electrophoresis

Microchip electrophoresis (MCE) has been advanced remarkably by the applications of several separation modes and the integration with several chemical operations on a single planer substrate. MCE shows superior analytical performance, e.g., high-speed analysis, high resolution, low consumption of reagents, and so on, whereas low-concentration sensitivity is still one of the major problems. To overcome this drawback, various online sample preconcentration techniques have been developed in MCE over the past 15 years, which have successfully enhanced the detection sensitivity in MCE. This review highlights recent developments in online sample preconcentration in MCE categorized on the basis of "dynamic" and "static" methods. The dynamic techniques including field amplified stacking, ITP, sweeping, and focusing have been easily applied to MCE, which provide effective enrichments of various analytes. The static techniques such as SPE and filtration have also been combined with MCE. In the static techniques, extremely high preconcentration efficiency can be obtained, compared to the dynamic methods. This review provides comprehensive tables listing the applications and sensitivity enhancement factors of these preconcentration techniques employed in MCE.     

Recent progress of on-line sample preconcentration techniques in microchip electrophoresis

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.     

Bias-free pneumatic sample injection in microchip electrophoresis

We have developed a new microfluidic chip capable of accurate metering, pneumatic sample injection, and subsequent electrophoretic separation. The pneumatic injection scheme, enabling us to introduce a solution without sampling bias unlike electrokinetic injection, is based upon the hydrophobicity and wettability of channel surfaces. An accurately metered solution of 10 nL could be injected by pneumatic pressure into a hydrophilic separation channel through Y-shaped hydrophobic valves, which consist of polydimethylsiloxane (PDMS) and fluorocarbon (FC) film layers. We demonstrated the successful pneumatic injection of a red ink solution into the separation channel as a proof of the concept. A mixture of fluorescein and dichlorofluorescein (DCF) could be baseline-separated using a single power source in microchip electrophoresis.     

Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.     

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis

Hydrostatic pressure sample injection method is able to minimize the number of electrodes needed for a microchip electrophoresis process; however, it neither can be applied for electrophoretic DNA sizing, nor can be implemented on the widely used single-cross microchip. This paper presents an injector design that makes the hydrostatic pressure sample injection method suitable for DNA sizing. By introducing an assistant channel into the normal double-cross injector, a rugged DNA sample plug suitable for sizing can be successfully formed within the cross area during the sample loading. This paper also demonstrates that the hydrostatic pressure sample injection can be performed in the single-cross microchip by controlling the radial position of the detection point in the separation channel. Rhodamine 123 and its derivative as model sample were successfully separated.     

Numerical simulation of DNA sample preconcentration in microdevice electrophoresis

A numerical model is presented for the accurate and efficient prediction of preconcentration and transport of DNA during sample introduction and injection in microcapillary electrophoresis. The model incorporates conservation laws for the different buffer ions, salt ions, and DNA sample, coupled through a Gaussian electric field to account for the field modifications that cause electromigration. The accuracy and efficiency required to capture the physics associated with such a complex transient problem are realized by the use of the finite element-flux corrected transport (FE-FCT) algorithm in two dimensions. The model has been employed for the prediction of DNA sample preconcentration and transport during electrophoresis in a double-T injector microdevice. To test its validity, the numerical results have been compared with the corresponding experimental data under similar conditions, and excellent agreement has been found. Finally, detailed results from a simulation of DNA sample preconcentration in electrophoretic microdevices are presented using as parameters the electric field strength and the other species concentrations. The effect of the Tris concentration on sample stacking is also investigated. These results demonstrate the great potential offered by the model for future optimization of such microchip devices with respect to significantly enhanced speed and resolution of sample separation.     

Integration of on-column immobilized enzyme reactor in microchip electrophoresis

A simple method integrating an immobilized enzyme reactor into a microchip electrophoresis device was developed. The enzyme immobilization into a microchip was performed by spotting and drying a drop of dissolved nitrocellulose (NC) on a glass substrate, and adsorbing enzyme on the reconstituted NC membrane. This enzyme-immobilized glass plate was assembled with a polydimethylsiloxane substrate on which the separation channel was fabricated. The advantage of this method is the ability to easily change the position and size of the reactor within the microchip electrophoresis device. A beta-galactosidase reaction was demonstrated with fluorescein di-beta-D-galactopyranoside using this integrated on-column enzyme reactor. A successful electrophoretic separation of its hydrolysis products, i.e., fluorescein mono-beta-D-galactopyranoside (FMG) and fluorescein, was achieved. Enzyme kinetics and inhibition of the beta-galactosidase using FMG and 2-phenylethyl beta-D-thiogalactoside, respectively, were also studied with microchip electrophoresis.     

Instrumentation design for hydrodynamic sample injection in microchip electrophoresis: A review

Reproducible and representative sample injection in microchip electrophoresis has been a bottleneck for quantitative analytical applications. Electrokinetic sample injection is the most used because it is easy to perform. However, this injection method is usually affected by sample composition and the bias effect. On the other hand, these drawbacks are overcome by the hydrodynamic (HD) sample injection, although this injection mode requires HD flow control. This review gives an overview of the basic principles, the instrumentation designs, and the performance of HD sample injection systems for microchip electrophoresis.     

Borate complexation-assisted field-enhanced sample injection for on-line preconcentration of cis-diol-containing compounds in capillary electrophoresis

A new on-line preconcentration technique called borate complexation-assisted field-enhanced sample injection (BCA-FESI) was proposed for preconcentrating cis-diol-containing compounds (CDCCs) in capillary electrophoresis (CE). The principle relies on amplification of the difference in the electrophoretic mobilities of CDCC in sample matrix and background electrolyte (BGE) through complexation of CDCC with borate in a sample matrix of basic pH and dissociation of the complex in a BGE of acidic pH. Meanwhile, CDCC and borate ions electro-injected into the capillary are finally in neutral state, which maintains the pre-filled low conductivity zone and thus allows for longer injection time. With catechol as a test compound, the principle and effectiveness of BCA-FESI was verified. As compared to conventional sample injection, BCA-FESI allowed for sensitivity enhancement of 1850-fold. The established method was further evaluated with three catechins, including (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), and (−)-epigallocatechin (EGC), in a standard mixture of trace content. The limit of detection (LOD) was found to be 1.4, 3.8, 17.5 nM (S/N = 3) for ECG, GCG, EGC, respectively. Finally, the BCA-FESI method was applied to a real sample of diluted tea beverage, in which the three catechins were detected.     

Sweeping and new on-line sample preconcentration techniques in capillary electrophoresis

Sweeping is a powerful on-line sample preconcentration technique that improves the concentration sensitivity of capillary electrophoresis (CE). This approach is designed to focus the analyte into narrow bands within the capillary, thereby increasing the sample volume that can be injected, without any loss of CE efficiency. It utilizes the interactions between an additive [i.e., a pseudostationary phase (PS) or complexing agent] in the separation buffer and the sample in a matrix that is devoid of the additive used. The accumulation occurs due to chromatographic partitioning, complexation or any interaction between analytes and the additive through electrophoresis. The extent of the preconcentration is dependent on the strength of interaction involved. Both charged and neutral analytes can be preconcentrated. Remarkable improvements—up to several thousandfold—in detection sensitivity have been achieved. This suggests that sweeping is a superior and general approach to on-line sample preconcentration in CE. The focusing mechanism of sweeping under different experimental conditions and its combination with other on-line preconcentration techniques are discussed in this review. The recently introduced techniques of transient trapping (tr-trapping) and analyte focusing by micelle collapse (AFMC) as well as other novel approaches to on-line sample preconcentration are also described.

On-line sample preconcentration in capillary electrophoresis. Fundamentals and applications

On-line preconcentration is one of the aspects of analytical method development using capillary electrophoretic techniques. The choice of the sample matrix alone can significantly alter both method sensitivity and separation efficiency. The recent trend to detect samples in narrower separation vessels also necessitates the need to improve detection sensitivity. The desire to detect very low levels of analytes using limited amounts of sample from biological specimens and the high separation efficiency obtainable using very large injections compared to classical small size injections also adds to this list. Indeed, one of the rich areas of research in the capillary electrophoresis field is on on-line sample preconcentration. More than 400 published research articles gathered from the http://www.webofscience.com from the year 2000 described a form of on-line preconcentration in capillary electrophoresis. This review provides a comprehensive table listing the applications of on-line preconcentration in capillary electrophoresis.     

Integrated isotachophoretic preconcentration with zone electrophoresis separation on a quartz microchip for UV detection of flavonoids

A quartz microchip integrated isotachophoretic (ITP) preconcentration with zone electrophoresis (ZE) separation was fabricated using a novel multi-point pressure method featured in normal temperature and lower pressure during bonding process. ITP followed by subsequential ZE of two flavonoids, quercetin and isorhamnetin on the microchip was performed consecutively on the homemade microfluidic workstation with UV detection, resulting in a decreased detectable concentration of 32-fold, compared to the ZE mode only, and their detection limits decreased down to 0.2 microg/mL and 1.2 microg/mL, respectively.     

Online sample pre-concentration via dynamic pH junction in capillary and microchip electrophoresis

Various analytical techniques have been developed over the years to analyse a large diversity of biomolecules with a constant push towards ultra-sensitive detection. CE is at the forefront of the most powerful analytical tools available to date when considering its superior efficiency and resolution; however, the technique suffers from poor sensitivity as a result of the short path length at the detection site and small injection volumes (typically <1% capillary length). One of the approaches to abate the inherent problem is to employ clever chemistry using sample focusing techniques whereby a large sample plug can be injected, preconcentrated and separated, producing excellent sensitivity and efficiency at the detector. This particular review will focus on the use of dynamic pH junction as a means of improving sensitivity in CE and focuses on the use of a change in analyte ionisation due to different pHs between the sample and electrolyte. The review provides a fundamental discussion of the mechanisms, buffer and sample conditions required to concentrate various analytes and a comprehensive list of published works in tabular format for easy identification of suitable conditions for new applications. The review further encompasses the use of dynamic pH junction in CE and its involvement in combination with other preconcentrations techniques to produce high sensitivity enhancements recorded between the years 1990-2010.     

Microchip electrophoresis of DNA following preconcentration at photopatterned gel membranes

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double-stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size-dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single-stranded oligonucleotide DNA (20-mer ssDNA). Baseline resolution of a six-band dye-labeled ladder with fragments 100-2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000-15,000 plates/cm, and detection limits as low as 1 pM per single dye-labeled fragment. The degree of preconcentration is tunable by at least 49-fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5-fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength.     

Integration of a carbon microelectrode with a microfabricated palladium decoupler for use in microchip capillary electrophoresis/electrochemistry

A method to integrate a carbon microelectrode with a microfabricated palladium decoupler for use in microchip capillary electrophoresis (CE) is detailed. As opposed to previous studies with decouplers for microchip CE, the working electrode material, which is made by micromolding of a carbon ink, is different from the decoupling electrode material (palladium). The manner in which the working electrode is made does not add additional etching or lithographic steps to the fabrication of the glass electrode plate. The hybrid poly(dimethylsiloxane)/glass device was characterized with fluorescence microscopy and by monitoring the CE-based separation of dopamine. Hydrodynamic voltammograms exhibited diffusion-limited currents occurring at potentials above +1.0 V. It was also shown that the half-wave potential does not shift as the separation potential is changed, as is the case in nondecoupled systems. Gated injections of dopamine in a 25 mM boric acid buffer (pH 9.2) showed a linear response from 200 to 5 microM (r2 = 0.9992), with a sensitivity of 5.47 pA/microM and an estimated limit of detection of 2.3 microM (0.621 fmol, S/N = 3). This is the first report of coupling a carbon electrode with a decoupler in microchip CE.     

Mini-electrochemical detector for microchip electrophoresis

This paper presents the development of a mini-electrochemical detector for microchip electrophoresis. The small size (3.6 x 5.0 cm2, W x L) of the detector is compatible with the dimension of the microchip. The use of universal serial bus (USB) ports facilitates installation and use of the detector, miniaturizes the detector, and makes it ideal for lab-on-a-chip applications. A fixed 10 M ohm feedback resistance was chosen to convert current of the working electrode to voltage with second gain of 1, 2, 4, 8, 16, 32, 64 and 128 for small signal detection instead of adopting selectable feedback resistance. Special attention has been paid to the power support circuitry and printed circuit board (PCB) design in order to obtain good performance in such a miniature size. The working electrode potential could be varied over a range of +/-2.5 V with a resolution of 0.01 mV. The detection current ranges from -0.3 x 10(-7) A to 2.5 x 10(-7) A and the noise is lower than 1 pA. The analytical performance of the new system was demonstrated by the detection of epinephrine using an integrated PDMS/glass microchip with detection limit of 2.1 microM (S/N = 3).     

On-line sample preconcentration by sweeping with dodecyltrimethylammonium bromide in capillary zone electrophoresis

On-line sample preconcentration of oligonucleotides with a new sweeping carrier was developed by using dodecyltrimethylammonium bromide (DTAB) below the critical micelle concentration (CMC). The sweeping results with DTAB below and above the CMC were compared. The use of DTAB below the CMC benefits the preconcentration of the oligonucleotides, while the use of DTAB above the CMC is good for hydrophobic small molecules. The factors affecting the sweeping results were optimized and this method was evaluated by constructing calibration curves for thrombin aptamers. The sweeping scheme produced a 112-fold sensitivity enhancement for the oligonucleotides relative to that run in a running buffer without DTAB. The sweeping method developed here can be a good reinforcement of the preconcentration scheme by sweeping when less-hydrophobic analytes or large negatively-charged molecules need to be preconcentrated.     

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross‐channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.     

Integrated multilayer microfluidic device with a nanoporous membrane interconnect for online coupling of solid-phase extraction to microchip electrophoresis

An integrated microfluidic device was developed for online coupling of solid-phase extraction to microchip electrophoresis (chip SPE-CE). With a nanoporous membrane sandwiched between two PDMS substrates, SPE preconcentration and electrophoretic separation can be carried out in upper and lower fluidic layers, separately and sequentially. During the SPE process, the thin membrane can act as a fluid isolator to prevent intermixing between two fluidic channels. However, when a pulse voltage is applied, the membrane becomes a gateable interconnect so that a small plug of concentrated analytes can be online injected into the lower channel for subsequent separations. This multilayer design provides a universal solution to online SPE-CE hyphenation. Both electroosmotic flow and hydrodynamic pumps have been adopted for SPE operation. SPE was performed on a 2.5 mm long microcolumn, with two weirs on both sides to retain the C(18)-coated silica beads. Rhodamine 123 and FITC-labelled ephedrine were used to test the operational performance of the hyphenation system. High separation efficiency and thousand-fold signal enhancement were achieved.     

On-line sample preconcentration of cationic analytes by dynamic pH junction in capillary electrophoresis

To improve detection sensitivity of cationic analytes, a dynamic pH junction technique was examined. Dynamic pH junction is an on-line focusing method in capillary electrophoresis (CE) based on the difference in the analyte's mobility between the background electrolyte (BGE) and sample matrix. The effects of pH values and concentrations of the BGE and the sample matrix on dynamic pH junction were examined. Optimization of analyte focusing resulted in enhanced detection responses of about 100-160-fold in terms of peak heights for some anilines in comparison to conventional injections. In particular, the concentration limits of detection (LOD) (S/N = 3) for the test anilines obtained with dynamic pH junction were from 1.9 to 3.7 ppb with UV detection without any pretreatment procedure.