Microchip electrophoresis separation of a panel of preterm birth biomarkers.

Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (µCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. µCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first µCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this µCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice.

Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (µCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a µCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined µCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and µCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

Integrated electrokinetically driven microfluidic devices with pH-mediated solid-phase extraction coupled to microchip electrophoresis for preterm birth biomarkers.

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low-abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH-mediated solid-phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed-phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50-fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15-fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.

Enantioseparation of tartaric acid by ligand-exchange capillary electrophoresis using contactless conductivity detection.

A method for the determination of tartaric acid enantiomers using CE with contactless conductivity detection has been developed. Cu(II) as a central metal ion together with L-hydroxyproline were used as a chiral selector, the BGE was composed of 7 mM CuCl2, 14 mM trans-4-hydroxy-L-proline, and 100 mM ε-aminocaproic acid; the pH was adjusted to 5 by hydrochloric acid. Separation with a resolution of 1.9 was achieved in 9 min in a polyacrylamide-coated capillary to suppress the EOF. Various counterions of the BGE were studied, and migration order reversal was achieved when switching from ε-aminocaproic acid to L-histidine. With detection limits of about 20 µM, the method was applied to the analysis of wine and grape samples; only L-tartaric acid was found.

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis.

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on-line preconcentration. 5 mmol.L⁻¹ naphthalene-1-sulfonic acid and 10 mmol.L⁻¹ triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large-volume sample stacking and the field-amplified sample injection were applied and compared. Large-volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L⁻¹, respectively. Field-amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L⁻¹, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid-phase extraction, which further improved LOD toward 5.6 × 10⁻¹° mol.L⁻¹ for PFOS and 6.4 × 10⁻¹° mol.L⁻¹ for PFOA and allows the method to be used for river water contamination screening or decomposition studies.

Multilabel hybridization probes for sequence-specific detection of sepsis-related drug resistance genes in plasmids.

Emerging antimicrobial drug resistance is increasing the complexity involved in treating critical conditions such as bacterial induced sepsis. Methods for diagnosing specific drug resistance tend to be rapid or sensitive, but not both. Detection methods like sequence-specific single-molecule analysis could address this concern if they could be adapted to work on smaller targets similar to those produced in traditional clinical situations. In this work we demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be created by immobilizing DNA with a biotin/streptavidin magnetic bead system, labeling with SYBR Gold, and rinsing the excess away while the probe retains multiple fluorophores. These probes with multiple fluorophores can then be used to label a bacterial plasmid target in a sequence-specific manner. These probes enabled the detection of 1 pM plasmid samples containing a portion of an antibiotic resistance gene sequence. This system shows the possibility of improving capture and fluorescence labeling of small nucleic acid fragments, generating lower limits of detection for clinically relevant samples while maintaining rapid processing times.

Analysis of trace degradation products (decarboxylated diastereoisomers) of S-adenosylmethionine by electrophoresis in capillaries with cationic coatings (N-methylpolyvinylpyridinium or divalent barium).

Commercial preparations of S-adenosylmethionine (SAM) when analyzed in uncoated capillaries show a minute impurity believed to be decarboxylated (dc) SAM. By using two types of cationic coatings, thus reducing the electro-endo-osmotic flow (EOF), it was possible to separate this impurity into two diastereoisomers of dcSAM. The coatings evaluated for this purpose were: (i) N-methylpolyvinylpyridinium, used under reversed EOF at acidic conditions (pH 4.0) and (ii) deposition of divalent barium at alkaline pH values (pH 9.4), providing reduced EOF. Under these conditions, it was possible to separate this impurity into two diastereoisomers, which by chemical synthesis were indeed proven to be dcSAM. It was further demonstrated that, in the alkylation of 5'-methylthioadenosine by 3-bromopropylamine in bromidric acid to dcSAM, another minute impurity was present, proven, via mass spectrometry, to consist of S-(5'-adenosyl)-3-thiopropylamine (decarboxylated and demethylated (dc-SAH)). The LOD for the two dcSAM diastereoisomers was assessed as 17.5 µg/mL and their LOQ as 25.5 µg/mL. By the barium-based protocol it was possible to quantify the dcSAM, present in a commercial sample of SAM, as a 0.1% impurity.

Assessment of CE for the identification of microorganisms.

The assessment of capillary electrophoresis for separation and identification of microorganisms is described in this article. The work brings a comparison of the application of uncoated capillaries modified with poly(ethylene oxide) and coated capillaries for the separation of model microorganisms Saccharomyces cerevisiae and Escherichia coli with Tris-borate-EDTA and Tris-citrate-fructose as electrolytes. The best separation was achieved in the coated capillary using 1 mM Tris-citrate-fructose buffer, pH 6.9. A simple identification based on the migration time and UV spectrum was found unsatisfactory and thus a method based on a post-separation cultivation and sequential analysis was developed. Off-line combination with mass spectrometric analysis with the use of desorption electrospray was shown to be an interesting alternative in a study of microorganisms. Mass spectra allowed distinguishing among analyzed cells.

Determination of some phenolic acids in Majorana hortensis by capillary electrophoresis with online electrokinetic preconcentration.

An online accumulation/mobilization preconcentration technique based on a dynamic pH junction technique and electrokinetic injection was employed for analysis of phenolic acids (sinapic, ferulic, coumarinic, caffeic, syringic, vanillic, and 4-hydroxybenzoic acid) in extracts from Majorana hortensis leaves. Samples were extracted by pressurized solvent extraction with acetone at 150 degrees C and 15 MPa. The capillary electrophoretic method employed 50 mmol.L (-1) sodium borate, pH 9.5, as the sample electrolyte, 50 mmol.L (-1) sodium phosphate, pH 2.5, as the background electrolyte, and 50 mmol.L (-1) sodium phosphate, pH 2.5, with 60 mmol.L (-1) sodium dodecyl sulfate as the mobilization electrolyte. The method allowed 720-fold to 5560-fold preconcentration of the phenolic acids during 30 min of electrokinetic accumulation with detection limits from 0.38 to 4.22 ng.mL (-1).

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection.

Capillary electrophoresis as a verification tool for immunochemical drug screening.

BACKGROUND: The aim of this work was to develop a simple capillary electrophoretic method as the verification and confirmation tool in the screening analysis for amphetamines, opiates, benzodiazepines and cocaine and their metabolites for toxicological applications. METHODS: 50 mM phosphate Tris pH 2.0 with 30% (v/v) of methanol was used as a background electrolyte that enabled fast separation of drugs and their metabolites in saliva and urine. Verification of the data from the electrophoretic method was done by High Performance Thin Layer Chromatography (HPTLC) and the immunochemical screening test QuikScreen. RESULTS: The experimental conditions of the Capillary Electrophoresis (CE) were partially optimized (mainly the influence of concentration and types of additives, e.g. cyclodextrines, organic solvents) and validated; the method was used for analysing samples from drug abusers. CONCLUSIONS: The non-instrumental, immunoassay tests could only confirm qualitative addictions and are mainly employed when the emergency detection of drugs is needed. For quantitative analysis and verification of obtained results the confirmation step is strongly recommended. The simple screening capillary zone electrophoresis method allows recognition of the most abused drugs. The agreement of the results from CE, HPTLC and QuikScreen test was more than 95%.

Sequence-specific DNA solid-phase extraction in an on-chip monolith: Towards detection of antibiotic resistance genes.

Antibiotic resistance of bacteria is a growing problem and presents a challenge for prompt treatment in patients with sepsis. Currently used methods rely on culturing or amplification; however, these steps are either time consuming or suffer from interference issues. A microfluidic device was made from black polypropylene, with a monolithic column modified with a capture oligonucleotide for sequence selective solid-phase extraction of a complementary target from a lysate sample. Porous properties of the monolith allow flow and hybridization of a target complementary to the probe immobilized on the column surface. Good flow-through properties enable extraction of a 100µL sample and elution of target DNA in 12min total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron-Mediated Metallo-ß-lactamase it was possible to extract and detect a 1pM sample with 83% recovery. Temperature-mediated elution by heating above the duplex melting point provides a clean extract without any agents that interfere with base pairing, allowing various labeling methods or further downstream processing of the eluent. Further integration of this extraction module with a system for isolation and lysis of bacteria from blood, as well as combining with single-molecule detection should allow rapid determination of antibiotic resistance.

Advances in monoliths and related porous materials for microfluidics.

In recent years, the use of monolithic porous polymers has seen significant growth. These materials present a highly useful support for various analytical and biochemical applications. Since their introduction, various approaches have been introduced to produce monoliths in a broad range of materials. Simple preparation has enabled their easy implementation in microchannels, extending the range of applications where microfluidics can be successfully utilized. This review summarizes progress regarding monoliths and related porous materials in the field of microfluidics between 2010 and 2015. Recent developments in monolith preparation, solid-phase extraction, separations, and catalysis are critically discussed. Finally, a brief overview of the use of these porous materials for analysis of subcellular and larger structures is given.

Analysis of Amphetamine-Derived Designer Drugs by Gas Chromatography with Mass Spectrometry.

The 22 amphetamine-derived synthetic drugs (ADSDs), mostly cathinones, were examined by gas chromatography with mass spectrometry using two different derivatization methods with (i) heptafluorobutyric anhydride (HFBA) and (ii) pentafluorobenzoyl chloride (PFBCl). Both developed derivatization approaches were evaluated and compared for urine and serum samples. Extraction procedures proved to give satisfactory results with regard to recoveries and extract purity, even though both derivatization methods reached acceptable sensitivity for the intended use. The derivatization with PFBCl showed better results with respect to retention and response stability, thus the PFBCl method was selected for validation. Calibration curves were linear over the tested concentration range of 20-1,000 ng/mL with the R(2) values ranging from 0.994 to 0.998. Intra- and interday precisions and accuracies were within 20% for all concentrations in the linear range. The limit of detection was determined to be lower than 2 ng/mL for all 22 analytes. The method proved to be a useful analytical tool in the course of systematic toxicological analysis.

Molecular imprinted polymeric porous layers in open tubular capillaries for chiral separations.

A new method has been developed for the preparation of molecular imprinted polymers as porous layers in open tubular (MIP-PLOT) capillary column formats for use in chiral separations by capillary liquid chromatography. The synthesis was based on 'in-capillary' ultraviolet (UV) initiated polymerization using light emitting diodes (LEDs) in conjunction with the continuous delivery of the pre-polymerization reagents into the polymerization zone of the capillary using an automated capillary delivery device. The relationships between exposure times, UV-light intensity and polymer layer thickness have been determined, as well as the effects of reagent delivery rate and multiple LED exposures on the layer thickness for various compositions of pre-polymerization mixtures. The polymer surface morphology was investigated by scanning electron microscopy (SEM). The non-steroidal anti-inflammatory drug S-ketoprofen was used as the template for the preparation of the MIP imprinted PLOT coatings. The separation performance with the ketoprofen racemate was investigated by capillary liquid chromatography. In contrast to alternative methods, which require the use of expensive chiral selectors, the described MIP PLOT stationary phases used non-chiral polymer precursors to create enantioselective nano-cavities through molecular self-assembly processes. The described fabrication methods provide a new avenue to tailor-make chiral MIP-PLOT capillary columns for the separation of chiral compounds present in complex or racemic analyte mixtures of chemical and biological origin.

Determination of brominated phenols in water samples by on-line coupled isotachophoresis with capillary zone electrophoresis.

A method for determination of nine brominated phenols as environmental risk compounds was developed by on-line coupled capillary isotachophoresis and capillary zone electrophoresis (ITP-CZE). For ITP step, 1x10(-2) mol L(-1) hydrochloric acid with 3x10(-2) mol L(-1) ammediol pH 9.1 was used as the leading electrolyte, and 3x10(-2) mol L(-1) beta-alanine with 2x10(-2) mol L(-1) sodium hydroxide pH 10.05 was used as the terminating electrolyte. As the background electrolyte for CZE separation, 2.5x10(-2) mol L(-1) beta-alanine with 2.5x10(-2) mol L(-1) lysine pH 9.6 was used. All electrolytes contained 0.05% or 0.1% (m/v) hydroxyethylcellulose to suppress the electroosmotic flow. UV detection at wavelength 220 nm was used. Detection limits in order of tens of nmol L(-1) were achieved. Good repeatability of migration times (less than 0.33% RSD) and good repeatability of peak areas (less than 7.19% RSD) at concentration level 5x10(-8) mol L(-1) were observed. Developed ITP-CZE method was applied to determination of brominated phenols in spiked tap and river water samples.

Electrokinetic partial filling technique as a powerful tool for enantiomeric separation of DL-lactic acid by CE with contactless conductivity detection.

A modified partial filling method for chiral separation of DL-lactic acid as the model chiral compound with vancomycin chloride as the chiral selector was developed by CE with contactless conductivity detection. Electrokinetic partial filling technique (EK-PFT) was used as an alternative method to the conventional hydrodynamic partial filling method. EK-PFT, in contrast to the hydrodynamic partial filling technique, allowed the removal of the chloride counterions from the chiral selector which otherwise led to poor sensitivity in conductivity detection. The baseline separation of DL-lactic acid as the model analyte was achieved in 5 min in a polyacrylamide-coated capillary. The best resolution was achieved by electrokinetic partial filling of vancomycin cations from the injection solution containing 5 mmol/L oxalate L-histidinium at pH 4.5 with 10 mmol/L vancomycin chloride. Computer simulation was used to explain the observed phenomena in the boundary between the inject vial and the capillary during the EK-PFT of vancomycin cations.