Sensitive detection of herbicide residues using field-amplified sample injection coupled with electrokinetic supercharging in flow-gated capillary electrophoresis.

Residues of glyphosate (GlyP) and its major degradation product, aminomethylphosphonic acid (AMPA), widely exist in the water system and plant products and thus are also present in the bodies of animals and humans. Although no solid evidence has been obtained, the concern about the cancer risk of GlyP is persistent. The measurement of GlyP and AMPA in trace levels is often needed but lacks readily available analytical approaches with detection sensitivity, accuracy and speed. This study aims to develop a simple and robust technique for the sensitive detection of GlyP and AMPA residues in a surface water system with flow-gated capillary electrophoresis (CE). Experimentally, water samples were first fluorogenically derivatized with 4-fluoro-7-nitrobenzofurazan (NBD-F) in a low-conductivity buffer at room temperature, and the mixture was injected and concentrated in the capillary based on field-amplified sample injection (FASI) coupled with electrokinetic supercharging (EKS). This scheme included a step of sample buffer injection upon electroosmotic pumping, where negatively charged analytes were electrophoretically rejected, followed by automatic voltage reversal for FASI-EKS. The detection sensitivity was improved by 296, 444, and 861 times for glufosinate (GluF), AMPA, and GlyP, respectively. The proposed method was validated in terms of accuracy, precision, limits of detection (LODs), and linearity. The LODs were estimated to be 50.0 pM, 5.0 pM, and 10.0 pM for GluF, AMPA, and GlyP, respectively. Its application was demonstrated by measuring GluF and AMPA in water samples collected from a local water system. This study provides an effective approach for the online preconcentration of negatively charged analytes, thus enabling the sensitive detection of herbicide residues in water samples. The method can also be applied to analyze other samples, including biological fluids and plant products, upon appropriate sample preparation such as solid phase extraction of analytes.

Rapid determination of free prolyl dipeptides and 4-hydroxyproline in urine using flow-gated capillary electrophoresis.

Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was > 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids. Graphical abstract Rapid quantitative analysis of prolyl dipeptides in urine using flow-gated capillary electrophoresis coupled with laser-induced fluorescence detection.

Alternate injections coupled with flow-gated capillary electrophoresis for rapid and accurate quantitative analysis of urine samples.

Capillary electrophoresis (CE) is a powerful separation technique with advantages over HPLC in terms of separation efficiency, speed, and cost. However, CE suffers in poor reproducibility in quantitative chemical analysis, which is one of major drawbacks preventing its widespread use in routine analytical laboratories. Here we report a novel strategy to enhance the quantitative capability of flow-gated CE. The platform integrated dual flow branches to respectively supply a sample and its standard additions that were then alternately injected into a single capillary for rapid separations (typically 20-90 s). A micro-fabricated switch was used to enable the alternate injections. It was assumed that the analytical system maintained constant conditions during neighboring injections that served as external self-standards for quantitation. This strategy was expected to reduce uncertainties caused by the fluctuation in capillary conditions and the drift of detection systems. Experimental results demonstrated that the dual-branch flow-gated CE coupled with alternate injections significantly improved the reproducibility with respect to peak height ratios under deliberate variations in injection volumes, separation voltages, optical focusing, and laser power; and thus the interday precision was ensured. To demonstrate its applicability, cyanide and amino acids in human urine were quantified rapidly with the one-point standard addition method after fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde (NDA), and the measurement accuracy was validated by determining the recovery of standard cyanide added to a urinary matrix. This strategy would be valuable to enable the quantitative capability of flow-gated CE in the measurements of a broad range of analytes, especially those lacking suited internal standards.

Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o-phthalaldehyde.

LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high-throughput analysis with flow-gated CE. Here, we report a fast fluorescein-labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow-gated CE. This scheme was based on the reaction between primary amines and o-phthalaldehyde in the presence of a fluorescent thiol, 2-((5-fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE-SH). The short reaction time (<30 s) was suited for on-line mixing and derivatization that was directly coupled with flow-gated CE for rapid electrophoretic separation and sensitive LIF detection. To maintain the effective concentration of reactive FACE-SH, Tris(2-carboxyethyl)phosphine was added to the derivatization reagents to prevent thiol loss due to oxidation. This labeling scheme was applied to the detection of neurotransmitters by coupling in vitro microdialysis with online derivatization and flow-gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on-chip labeling of proteins retained on support in SPE.

On-line preconcentration of fluorescent derivatives of catecholamines in cerebrospinal fluid using flow-gated capillary electrophoresis.

Flow-gated capillary electrophoresis (CE) coupled with microdialysis has become an important tool for in vivo bioanalytical measurements because it is capable of performing rapid and efficient separations of complex biological mixtures thus enabling high temporal resolution in chemical monitoring. However, the limit of detection (LOD) is often limited to a micro- or nano-molar range while many important target analytes have picomolar or sub-nanomolar levels in brain and other tissues. To enhance the capability of flow-gated CE for catecholamine detection, a novel and simple on-line sample preconcentration method was developed exclusively for fluorescent derivatives of catecholamines that were fluorogenically derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The effective preconcentration coupled with the sensitive laser-induced fluorescence (LIF) detection lowered the LOD down to 20pM for norepinephrine (NE) and 50pM for dopamine (DA) at 3-fold of S/N ratio, and the signal enhancement was estimated to be over 100-fold relative to normal injection when standard analytes were dissolved in artificial cerebrospinal fluid (aCSF). The basic focusing principle is novel since the sample plug contains borate while the background electrolyte (BGE) is void of borate. This strategy took advantage of the complexation between diols and borate, through which one negative charge was added to the complex entity. The sample derivatization mixture was electrokinetically injected into a capillary via the flow-gated injection, and then NE and DA derivatives were selectively focused to a narrow zone by the reversible complexation. Separation of NE and DA derivatives was executed by incoming surfactants of cholate and deoxycholate mixed in the front BGE plug. This on-line preconcentration method was finally applied to the detection of DA in rat cerebrospinal fluid (CSF) via microdialysis and on-line derivatization. It is anticipated that the method would be valuable for in vivo monitoring of DA and NE in various brain regions of live animals on flow-gated CE or microchip platforms.

A direct and rapid method to determine cyanide in urine by capillary electrophoresis.

Cyanides are poisonous chemicals that widely exist in nature and industrial processes as well as accidental fires. Rapid and accurate determination of cyanide exposure would facilitate forensic investigation, medical diagnosis, and chronic cyanide monitoring. Here, a rapid and direct method was developed for the determination of cyanide ions in urinary samples. This technique was based on an integrated capillary electrophoresis system coupled with laser-induced fluorescence (LIF) detection. Cyanide ions were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and a primary amine (glycine) for LIF detection. Three separate reagents, NDA, glycine, and cyanide sample, were mixed online, which secured uniform conditions between samples for cyanide derivatization and reduced the risk of precipitation formation of mixtures. Conditions were optimized; the derivatization was completed in 2-4min, and the separation was observed in 25s. The limit of detection (LOD) was 4.0nM at 3-fold signal-to-noise ratio for standard cyanide in buffer. The cyanide levels in urine samples from smokers and non-smokers were determined by using the method of standard addition, which demonstrated significant difference of cyanide levels in urinary samples from the two groups of people. The developed method was rapid and accurate, and is anticipated to be applicable to cyanide detection in waste water with appropriate modification.

Prototyping of poly(dimethylsiloxane) interfaces for flow gating, reagent mixing, and tubing connection in capillary electrophoresis.

Integrated microfluidic systems coupled with electrophoretic separations have broad application in biologic and chemical analysis. Interfaces for the connection of various functional parts play a major role in the performance of a system. Here, we developed a rapid prototyping method to fabricate monolithic poly(dimethylsiloxane) (PDMS) interfaces for flow-gated injection, online reagent mixing, and tube-to-tube connection in an integrated capillary electrophoresis (CE) system. The basic idea was based on the properties of PDMS: elasticity, transparency, and suitability for prototyping. The molds for these interfaces were prepared by using commercially available stainless steel wires and nylon lines or silica capillaries. A steel wire was inserted through the diameter of a nylon line and a cross format was obtained as the mold for PDMS casting of flow gates and 4-way mixers. These interfaces accommodated tubing connection through PDMS elasticity and provided easy visual trouble shooting. The flow gate used smaller channel diameters, thus reducing flow rate by 25-fold for effective gating compared with mechanically machined counterparts. Both PDMS mixers and the tube-to-tube connectors could minimize the sample dead volume by using an appropriate capillary configuration. As a whole, the prototyped PDMS interfaces are reusable, inexpensive, convenient for connection, and robust when integrated with the CE detection system. Therefore, these interfaces could see potential applications in CE and CE-coupled systems.

Nanofluidics in chemical analysis.

Nanofluidic architectures and devices have already had a major impact on forefront problems in chemical analysis, especially those involving mass-limited samples. This critical review begins with a discussion of the fundamental flow physics that distinguishes nanoscale structures from their larger microscale analogs, especially the concentration polarization that develops at nanofluidic/microfluidic interfaces. Chemical manipulations in nanopores include nanopore-mediated separations, microsensors, especially resistive-pulse sensing of biomacromolecules, fluidic circuit analogs and single molecule measurements. Coupling nanofluidic structures to three-dimensional microfluidic networks is especially powerful and results in applications in sample preconcentration, nanofluidic injection/collection and fast diffusive mixing (160 references).

Multidimensional separation of chiral amino acid mixtures in a multilayered three-dimensional hybrid microfluidic/nanofluidic device.

Microscale total analysis systems (microTAS) allow high-throughput analyses by integrating multiple processes, parallelization, and automation. Here we combine unit operations of microTAS to create a device that can perform multidimensional separations using a three-dimensional hybrid microfluidic/nanofluidic device composed of alternating layers of patterned poly(methyl methacrylate) and nanocapillary array membranes constructed from nuclear track-etched polycarbonate. Two consecutive electrophoretic separations are performed, the first being an achiral separation followed by a chiral separation of a selected analyte band. Separation conditions are optimized for a racemic mixture of fluorescein-isothiocyanate-labeled amino acids, serine and aspartic acid, chosen because there are endogenous D-forms of these amino acids in animals. The chiral separation is implemented using micellar electrokinetic chromatography using beta-cyclodextrin as the chiral selector and sodium taurocholate as the micelle-forming agent. Analyte separation is monitored by dual-beam laser-induced fluorescence detection. After separation in the first electrophoretic channel, the preselected analyte is sampled by the second-stage separation using an automated collection sequence with a zero-crossing algorithm. The controlled fluidic environment inherent to the three-dimensional architecture enables a series of separations in varying fluidic environments and allows sample stacking via different background electrolyte pH conditions. The ability to interface sequential separations, selected analyte capture, and other fluidic manipulations in the third dimension significantly improves the functionality of multilayer microfluidic devices.

Centrifugal sedimentation for selectively packing channels with silica microbeads in three-dimensional micro/nanofluidic devices.

Incorporation of nanofluidic elements into microfluidic channels is one approach for adding filtration and partition functionality to planar microfluidic devices, as well as providing enhanced biomolecular separations. Here we introduce a strategy to pack microfluidic channels with silica nanoparticles and microbeads, thereby indirectly producing functional nanostructures; the method allows selected channels to be packed, here demonstrated so that a separation channel is packed while keeping an injection channel unpacked. A nanocapillary array membrane is integrated between two patterned microfluidic channels that cross each other in vertically separated layers. The membrane serves both as a frit for bead packing and as a fluid communication conduit between microfluidic channels. Centrifugal force-assisted sedimentation is then used to selectively pack the microfluidic channels using an aqueous silica bead suspension loaded into the appropriate inlet reservoirs. This packing approach may be used to simultaneously pack multiple channels with silica microbeads having different sizes and surface properties. The chip design and packing method introduced here are suitable for packing silica particles in sizes ranging from nanometers to micrometers and allow rapid (approximately 10 min) packing with high quality. The liquid/analyte transport characteristics of these packed micro/nanofluidic devices have potential utility in a wide range of applications, including electroosmotic pumping, liquid chromatographic separations, and electrochromatography.

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection.

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.

Protein-aptamer binding studies using microchip affinity capillary electrophoresis.

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.

Fluidic communication between multiple vertically segregated microfluidic channels connected by nanocapillary array membranes.

Hybrid microfluidic/nanofluidic devices offer unique capabilities for manipulating and analyzing minute volumes of expensive or hard-to-obtain samples. Here, multilayer poly-(methyl methacrylate) microchips, with multiple spatially isolated microfluidic channels interconnected by nanocapillary array membranes (NCAMs), are fabricated using an adhesive contact printing process. The NCAMs, positioned between the microfluidic channel layers, add functionality to the inter-microchannel fluid transfer unit operation. They do so because the transport of specific analytes through the NCAM can be controlled by adjusting the ionic strength, the polarity of the applied bias, the surface charge density, and the pore size. A simplified, floating injection technique for NCAM-coupled nanofluidic devices is described and compared with conventional biased injection. In the floating injection approach, a voltage is applied across the injection channel and the slight electric field extension at the cross-section is used to transfer analytes through the nanopores to the separation channel. Floating injection excels in plug reproducibility, separation resolution, and operation simplicity, although it decreases assay throughput relative to biased injection. Floating injection can avoid the uneven distribution of analytes in the microfluidic channel that sometimes results from biased injection because of the volume mismatch between NCAM nanopore transport capacity and the supply of fluid. Moreover, the pressure-driven flow caused by the mismatch of the EOFs in the microfluidic channels connected by an NCAM must be considered when using NCAMs with pore diameters below 50 nm.

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis.

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.

Study of injection bias in a simple hydrodynamic injection in microchip CE.

The electrokinetically pinched method is the most commonly used mode for sample injection in microchip capillary electrophoresis (microCE) due to its simplicity and well-defined sample volume. However, the limited injection volume and the electrophoretic bias of the pinched injection may limit its universal usage to specific applications. Several hydrodynamic injection methods in microCE have been reported; however, almost all claimed that their methods are bias-free without considering the dispensing bias. To investigate the dispensing bias, a simple hydrodynamic injection was developed in single-T and double-T glass microchips. The sample flow was produced by hydrostatic pressure generated by the liquid level difference between the sample reservoir and the other reservoirs. The reproducibility of peak area and peak area ratio was improved to a significant extent using large-surface reservoirs for the buffer reservoir and the sample waste reservoir to reduce the Laplace pressure effect. Without a voltage applied on the sample solution, the voltage-related sample bias was eliminated. The dispensing bias was analyzed theoretically and studied experimentally. It was demonstrated that the dispensing bias existed and could be reduced significantly by appropriately setting up the voltage configuration and by controlling the appropriate liquid level difference.

Frontal analysis in microchip CE: a simple and accurate method for determination of protein-DNA dissociation constant.

Equilibrium constants, such as the dissociation constant (K(d)), are a key measurement of noncovalent interactions that are of importance for the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate CE method for the determination of K(d). Microchip CE coupled with LIF detection was used to determine K(d) of protein-DNA interactions using the FA method. A model system of IgE and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K(d) of the IgE-aptamer pair was determined as 6 +/- 2 nM which is consistent with the reported results (8 nM).

Unlimited-volume electrokinetic stacking injection in sweeping capillary electrophoresis using a cationic surfactant.

Sweeping is an effective and convenient way for online sample preconcentration in micellar electrokinetic chromatography. The usual procedure includes a hydrodynamic injection step carried out by applying pressure to the sample vial followed by the subsequent sweeping and separation processes. The injected sample volume is limited by the dimensions of the capillary because a part of the capillary has to be left free of sample solution for the subsequent sweeping and separation steps. In addition, when a short capillary, such as 4-10 cm, is used for sweeping, the injected sample volume is small even if the entire capillary is filled with sample solution. To solve this problem, an electrokinetic stacking injection (EKSI) scheme was developed by using a cationic surfactant, dodecyltrimethylammonium bromide, for sweeping in capillary electrophoresis. An experimental model was proposed, and the entire process was theoretically analyzed. According to the theoretical discussion, the optimal conditions for two model analytes, 5-carboxyfluorescein (5-FAM) and sodium fluorescein (FL), were experimentally determined. The injected sample plug lengths for 5-FAM and FL under 20.1 kV for 60 min were experimentally estimated as 836 and 729 cm, corresponding to 28- and 24-fold the effective capillary length, respectively. The EKSI scheme resulted in increased detection factors for 5-FAM and FL of 4.5 x 10(3) and 4.0 x 10(3) using 60-min injection relative to a traditional pressure injection.

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.

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis.

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.