Thread-powered cell lysis and isotachophoresis: unlocking microbial DNA for diverse molecular applications.

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection.

Transient isotachophoresis-Capillary zone electrophoresis-Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids.

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76-100 % for urine, 12-54 % for serum, 21-106 % for plasma, and 25-98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

Sorting and simultaneous quantitation of intact mixed-cell samples via capillary isotachophoresis.

A great need currently exists for rapid, inexpensive, and accurate methods for microbial analysis in the medical, food, industrial, and water quality fields. Here, a novel capillary isotachophoresis (CITP) method is presented for the focusing, sorting, and quantitation of intact cells in mixed samples based on their electrophoretic mobility ranges. Using a series of ion spacers dissolved in the sample, this technique results in several efficient cell peaks in the electropherogram corresponding to specific cell electrophoretic mobility ranges. The concentrations of different species in mixed-cell samples are determined from the cell peak areas and the known peak response factors for the cell species using a series of linear equations. Method design and optimization are discussed, including the choice of running buffer, pH, and ion spacers. Mixed-cell samples of up to four different species were focused and quantified as a proof-of-principle of the method. When sample cell concentrations were toward the middle of the linear response range, accuracies between 1% and 11% and relative standard deviations of 1%-14% were obtained, depending on the number of cell species in the mixture. This work provides a useful basis for future studies of cell quantitation using CITP, which could be potentially applied to a variety of fields including cell growth studies, microbial contamination testing, and sterility testing.

Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis.

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.

Microfluidic paper and thread-based separations: Chromatography and electrophoresis.

Paper and thread are widely used as the substrates for fabricating low-cost, disposable, and portable microfluidic analytical devices used in clinical, environmental, and food safety monitoring. Concerning separation methods including chromatography and electrophoresis, these substrates provide unique platforms for developing portable devices. This review focuses on summarizing recent research on the miniaturization of the separation techniques using paper and thread. Preconcentration, purification, desalination, and separation of various analytes are achievable using electrophoresis and chromatography methods integrated with modified or unmodified paper/thread wicking channels. A variety of 2D and 3D designs of paper/thread platforms for zone electrophoresis, capillary electrophoresis, and modified/unmodified chromatography are discussed with emphasis on their limitation and improvements. The current progress in the signal amplification strategies such as isoelectric focusing, isotachophoresis, ion concentration polarization, isoelectric focusing, and stacking methods in paper-based devices are reviewed. Different strategies for chromatographic separations based on paper/thread will be explained. The separation of target species from complex samples and their determination by integration with other analytical methods like spectroscopy and electrochemistry are well-listed. Furthermore, the innovations for plasma and cell separation from blood as an important human biofluid are presented, and the related paper/thread modification methods are explored.

Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.

Developed and Validated Capillary Isotachophoresis Method for the Rapid Determining Organic Acids in Children's Saliva.

One of the current challenges facing researchers is the search for alternative biological material, as opposed to routinely and invasively collected (such as blood), as the analysis of the former would provide information about the state of human health, allowing for the diagnosis of diseases in their early stages. With the search for disease biomarkers in alternative materials, the development of newer analytical solutions has been observed. This study aims to develop a reliable analytical method using the capillary isotachophoresis technique for the determination of organic acids in children's saliva, the presence/elevation of which can be used in the future for diagnostic purposes. Organic acids such as formic, lactic, acetic, propionic, and butyric acid, were determined in the saliva of healthy children without carious lesions. The limit of quantification determined in the validation process was found to vary from 0.05 to 1.56 mg/L, the recoveries at the two levels were determined to vary between 90% and 110% for level I, while for level II the corresponding values of 75% and 106% were found; the presentation, expressed as relative standard deviation values (RSD), did not exceed 5%. The parameters determined while validating the results method indicated that the obtained are reliable. The Red-Green-Blue (RGB) additive color model was used for the evaluation of the method. This comparative analysis allowed us to define the color of the method, which expresses whether it meets the given assumptions and requirements. According to the RGB model, the isotachophoresis method developed requires less reagent input, shorter sample preparation times, and results with lower energy consumption. Thus, the subject procedure may provide an alternative, routine tool for determining organic acids in human saliva, to be applied in the diagnosing of diseases of various etiological origins.

Evolution of the theoretical description of the isoelectric focusing experiment: I. The path from Svensson's steady-state model to the current two-stage model of isoelectric focusing.

In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.

Evolution of the theoretical description of the isoelectric focusing experiment: II. An open system isoelectric focusing experiment is a transient, bidirectional isotachophoretic experiment.

The carrier ampholytes-based (CA-based) isoelectric focusing (IEF) experiment evolved from Svensson's closed system IEF (constant spatial current density, absence of convective mixing, counter-balancing electrophoretic and diffusive fluxes yielding a steady state pH gradient) to the contemporary open system IEF (absence of convective mixing, large cross-sectional area electrode vessels, lack of counter-balancing electrophoretic- and diffusive fluxes leading to transient pH gradients). Open system IEF currently is described by a two-stage model: In the first stage, a rapid IEF process forms the pH gradient which, in the second stage, is slowly degraded by isotachophoretic processes that move the most acidic and most basic CAs into the electrode vessels. An analysis of the effective mobilities and the effective mobility to conductivity ratios of the anolyte, catholyte, and the CAs indicates that in open system IEF experiments a single process, transient bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on until it is turned off. In tbdITP, the anolyte and catholyte provide the leading ions and the pI 7 CA or the reactive boundary of the counter-migrating H3 O+ and OH- ions serves as the shared terminator. The outcome of the tbdITP process is determined by the ionic mobilities, pKa values, and loaded amounts of all ionic and ionizable components: It is constrained by both the transmitted amount of charge and the migration space available for the leading ions. tbdITP and the resulting pH gradient can never reach steady state with respect to the spatial coordinate of the separation channel.

Evolution of the theoretical description of the isoelectric focusing experiment: III. Carrier ampholyte behavior in transient, bidirectional isotachophoresis.

In modern isoelectric focusing (IEF) systems, where (i) convective mixing is prevented by gels or small cross-sectional area separation channels, (ii) current densities vary spatially due to the presence of electrode vessels with much larger cross-sectional areas than those of the gels or separation channels, and (iii) electrophoretic and diffusive fluxes do not balance each other, stationary, steady-state pH gradients cannot form (open-system IEF). Open-system IEF is currently described as a two-stage process: A rapid IEF process forms the pH gradient from the carrier ampholytes (CAs) in the first stage, then isotachophoretic processes degrade the pH gradient in the second stage as the extreme pI CAs are moved into the electrode vessels where they become diluted. Based on the ratios of the local effective mobilities and the local conductivities ( µ L eff ( x ) $\mu _{\rm{L}}^{{\rm{eff}}}( x )$ / κ ( x ) $\kappa ( x )$ values) of the anolyte, catholyte, and the CAs, we pointed out in the preceding paper (Vigh G, Gas B, Electrophoresis 2023, 44, 675-88) that in open-system IEF, a single process, transient, bidirectional isotachophoresis (tbdITP) operates from the moment current is turned on. In this paper, we demonstrate some of the operational features of the tbdITP model using the new ITP/IEF version of Simul 6.

Free-flow biomolecular concentration and separation of proteins and nucleic acids using teíchophoresis.

The ability to preconcentrate, separate, and purify biomolecules, such as proteins and nucleic acids, is an important requirement for the next generation of portable diagnostic tools for environmental monitoring and disease detection. Traditionally, such pretreatment has been accomplished using large, centralized liquid- or solid-phase extraction equipment, which can be time-consuming and requires many processing steps. Here, we present a newly developed electrokinetic concentration technique, teíchophoresis (TPE), to concentrate and separate proteins, and to concentrate nucleic acids. In TPE, a free-flowing sample is exposed to a perpendicular electric field in the vicinity of a mass-impermeable conductive wall and a conductive terminating electrolyte (TE), which creates a high electric field strength zone between the lower mobility sample and the no-flux barrier. Unlike a similar electrokinetic concentration method, isotachophoresis (ITP), TPE does not require a leading electrolyte (LE), yet still enables a continuous field-driven electrophoretic ion migration across the channel and a free-flowing biomolecular concentration at the conductive wall. Here, we demonstrate the use of free-flow TPE (FFTPE) to manipulate biomolecular samples containing proteins or nucleic acids. We first use TPE to drive a 6.6-fold concentration increase of avidin-FITC, and also demonstrate protein separation and stacking between ovalbumin-fluorescein and BSA-AlexaFluor 555, both without the use of a conventional LE. Further, we utilize TPE to perform a 21-fold concentration increase of nucleic acids. Our results show that TPE is biocompatible with both proteins and nucleic acids, requires only 10 V DC, produces no significant sample pH changes during operation, and demonstrates that this method can be used as an effective sample pretreatment to prepare biological samples for downstream analysis in a continuous free-flowing microfluidic channel.

Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis.

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution. Thermal gels were employed as a separations matrix because of their unique ability to change viscosity in response to temperature. Proteins were added into thermal gel and loaded into a microfluidic device. Electrolyte optimization was conducted to achieve robust tITP to isotachophoretically preconcentrate proteins and then electrophoretically separate them. Electropherograms were collected through both time and distance to enable both small and large proteins to be measured within a single analysis. The effects of temperature were evaluated and found to exhibit a pronounced effect on the separation. Temperature gradients were then employed to alter thermal gel viscosity over time to maximize separation resolution between proteins. The results herein demonstrate how gradient TG-tITP achieves rapid, high-performance separations of native proteins. This analysis provided a wide mass range (6-464 kDa) with two-fold higher resolution than native PAGE while requiring 15,000-fold less protein loading and providing five-fold faster analysis times.

Preparative protein concentration from biofluids by epitachophoresis.

With increasing demands on protein analyses in complex biological matrices, the insistence on developing new sample preparation techniques is rising. Recently, we introduced a new displacement electrophoresis technique (epitachophoresis) and instrumentation for preparative concentration and cleaning of DNA samples. This work describes the possibility of applying this device to protein samples. We have developed a method for the epitachophoretic concentration of proteins in a cationic mode and tested it by concentrating and collecting the protein zones from complex biological matrices (urine and growth medium). Under optimized conditions, we have obtained recoveries up to 99%. Furthermore, the applicability of the developed method was proven by concentrating and collecting the cytochrome c zone from a HeLa cell line growth medium, where the protein cytochrome c was released during cell apoptosis.

Induced sample via transient isotachophoresis mediated with sweeping in micellar electrokinetic chromatography for the dual-stacking strategy of non-steroidal anti-inflammatory drugs in environmental water samples.

Realising the need to devise a simple, sensitive, and reliable detection method, this study investigated the development of a dual-stacking transient isotachophoresis (t-ITP) and sweeping in micellar electrokinetic chromatography with diode array detector (t-ITP/sweeping-MEKC-DAD) for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs); ketoprofen, diclofenac and naproxen from aqueous matrices. Prior to the system setup, various parameters were optimised to assess the potential use of the t-ITP paired with the sweeping stacking technique in micellar background electrolyte for dual preconcentration and separation of trace amounts of NSAIDs. Once the optimum conditions have been established, the method performance was validated and applied to 17 environmental water samples. Based on the results, the combined t-ITP and sweeping approach significantly improved the stacking and separation sensitivity. A large volume of samples could also be introduced and subsequently separated by MEKC with greater focusing effects due to the sweeping. Under optimised conditions, the developed method exhibited excellent linearity at a high range (0.1-500 ng/mL, r2 ≥ 0.998), low limits of detection (LODs) of 0.01-0.07 ng/mL, and a remarkable relative recovery (RR) of 99.6-101.9% with a relative standard deviation (RSD) of 1.4-8.6% (n = 9). Ultimately, the sensitivity enhancement factors improved up to 666-fold using the optimised method. Therefore, the proposed method presents a simplified yet effective and suitable for the determination of NSAIDs from aqueous matrices.

Continuous Molecular Concentration and Separation Using Pulsed-Field Conductive-Wall Single-Buffer Teíchophoresis.

We present an experimental study of a novel continuous electrokinetic molecular concentration and separation technique termed teíchophoresis (TPE). We demonstrate here that TPE can serve as a potential alternative to the electrokinetic method isotachophoresis (ITP). In ITP, an electric field serves to focus charged species between a low-mobility terminating electrolyte (TE) and a high-mobility leading electrolyte (LE). Similarly, TPE serves to focus charged species between a low-mobility TE; however, the LE is conveniently replaced with a no-flux boundary generated by a conductive wall. The electric field can still penetrate this no-flux region due to the wall's finite conductivity, but ion migration is impeded due to the physicality of the wall. We perform detailed concentration and separation experiments across varying electric potentials, flow rates, and TE concentrations. We also show that TPE can achieve a 60,000-fold concentration factor continuously without an LE, using only 10 V DC. In comparison with conventional batch-driven ITP, continuous free-flow wall TPE (FFTPE) has the potential to serve as a simplified alternative method. FFTPE offers a high concentration power at a fraction of the required voltage, does not require an LE, and has the increased throughput potential of a continuous process.

Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH 8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine ß-lactoglobulin A.

Picomolar detection limits for glyphosate by two-dimensional column-coupled isotachophoresis/capillary zone electrophoresis-mass spectrometry.

Capillary electrophoresis-mass spectrometry often lacks sufficient limits of detection for trace substances in the environment due to its low loadability. To overcome this problem, we conducted a feasibility study for column-coupling isotachophoresis to capillary electrophoresis-mass spectrometry. The first dimension isotachophoresis preconcentrated the analytes. The column-coupling of both dimensions was achieved by a hybrid capillary microfluidic chip setup. Reliable analyte transfer by voltage switching was enabled by an in-chip capacitively coupled contactless conductivity detector placed around the channel of the common section between two T-shaped crossings in the chip connecting both dimensions. This eliminated the need to calculate the moment of analyte transfer. A commercial capillary electrophoresis-mass spectrometry instrument with easily installable adaptations operated the setup. Prior to coupling isotachophoresis with capillary zone electrophoresis-mass spectrometry, both dimensions were optimized individually by simulations and verified experimentally. Both dimensions were able to stack/separate all degradation products of glyphosate, the most important herbicide applied worldwide. The first dimension isotachophoresis also removed phosphate, which is a critical matrix component in many environmental samples. Enrichment and separation of glyphosate and its main degradation product aminomethylphosphonic acid by the two-dimensional setup provided an excellent limit of detection of 150 pM (25 ng/L) for glyphosate.

Recent progress in analytical capillary isotachophoresis (2018 - March 2022).

This review brings a survey of papers on analytical capillary and microchip isotachophoresis published since 2018 until the first quarter of 2022. Theoretical papers extending fundamental knowledge include those on computer simulations that remain an important research tool useful in the design of electrolyte systems. Many papers are focused on instrumental aspects where new media including microfluidic devices and their hyphenation to various detection techniques bring remarkable results. Papers reporting analytical applications demonstrate the potential of contemporary analytical isotachophoresis. Although it is not being used on a mass scale, its special features are attracting continued interest resulting in applications of isotachophoresis both as a stand-alone analytical method and as a part of multidimensional separation techniques.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Trace determination of perchlorate in drinking water by capillary zone electrophoresis with isotachophoresis sample cleanup and conductivity detection.

An analytical test procedure for the direct determination of trace levels of perchlorate in drinking water by isotachophoresis combined with capillary zone electrophoresis was developed. A capillary electrophoresis analyzer with column coupling technology, capable of combining capillaries with different internal diameters, was employed in combination with conductivity detection. This combination of the capillary electrophoresis techniques facilitated preconcentration of the trace analytes and elimination of potentially interfering macro-components. To eliminate the influence of weak and moderately strong acids on the migration of perchlorate, acidic leading electrolyte (pH 3.2) in the isotachophoresis step and acidic background electrolyte (pH 3.9) in the zone electrophoresis step were chosen. The addition of polyvinylpyrrolidone into the electrolytes enhanced the resolution of perchlorate from other anions, especially remaining anionic macro-components. The developed method is characterized by good repeatability of migration time (relative standard deviation less than 0.2%) as well as peak area (relative standard deviation less than 5.9%), linearity (R = 0.9996), recoveries (100-112%), and sample throughput (90 samples/24 h). The limit of quantitation for perchlorate in drinking water was achieved at 12.5 nmol/L (1.25 µg/L). This approach is more sensitive and more robust than transient isotachophoresis and offers advantages over some more established analytical techniques such as ion chromatography.