Investigation of interactions between biological thiols and gold nanoparticles by capillary electrophoresis with laser-induced fluorescence.

Biological thiols spontaneously form a stable Au-S dative bond with gold nanoparticles (AuNP) that might be used for their selective extraction and enrichment in biological samples. In this work, interactions of selected biological thiols (glutathione, cysteine, homocysteine [Hcys], cysteamine [CA], and N-acetylcysteine) with AuNP stabilized by different capping agents (citrate, Tween 20, Brij 35, CTAB, SDS) were investigated by UV-Vis spectroscopy and capillary electrophoresis with laser-induced fluorescence. Spectrophotometric measurements showed aggregation of Hcys and CA with AuNP. In contrast, it was confirmed by CE-LIF that biological thiols were adsorbed to all types of AuNP. Citrate-capped AuNP were selected for AuNP-based extraction of biological thiols from exhaled breath condensate (EBC). Dithiothreitol was utilized for desorption of biological thiols from the AuNP surface, which was followed by derivatization with eosin-5-maleimide and CE-LIF analysis. AuNP-based extraction increased the sensitivity of CE-LIF analysis; however, further optimization of methodology is necessary for accurate quantification of biological thiols in EBC.

Use of metal nanoparticles for preconcentration and analysis of biological thiols.

Metal nanoparticles (NPs) exhibit several unique physicochemical properties, including redox activity, surface plasmon resonance, ability to quench fluorescence, biocompatibility, or a high surface-to-volume ratio. They are being increasingly used in analysis and preconcentration of thiol containing compounds, because they are able to spontaneously form a stable Au/Ag/Cu-S dative bond. They thus find wide application in environmental and particularly in medical science, especially in the analysis of biological thiols, the endogenous compounds that play a significant role in many biological systems. In this review article, we provide an overview of various types of NPs that have been applied in analysis and preconcentration of biological thiols, mainly in human biological fluids. We first discuss shortly the types of NPs and their synthesis, properties, and their ability to interact with thiol compounds. Then we outline the sample preconcentration and analysis methods that were used for this purpose with special emphasis on optical, electrochemical, and separation techniques.

Analysis of bile acids in human biological samples by microcolumn separation techniques: A review.

Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle of the twentieth century, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. This article overviews the literature from the last two decades (2000-2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, used in their analysis. This is followed by a discussion on selected clinical applications.

Development of CE-C4D Method for Determination Tropane Alkaloids.

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C4D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) ß-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters-precision (CV): 1.07-2.08%, accuracy of the assay (recovery, RE): 101.0-102.7% and matrix effect (ME): 92.99-94.23%. Moreover, the optimized CE-C4D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.

Sub-Minute Analysis of Lactate from a Single Blood Drop Using Capillary Electrophoresis with Contactless Conductivity Detection in Monitoring of Athlete Performance.

A simple and fast method for the analysis of lactate from a single drop of blood was developed. The finger-prick whole blood sample (10 µL) was diluted (1:20) with a 7% (w/v) solution of [tris(hydroxymethyl)methylamino] propanesulfonic acid and applied to a blood plasma separation device. The device accommodates a membrane sandwich composed of an asymmetric polysulfone membrane and a supporting textile membrane that allows the collection of blood plasma into a narrow glass capillary in less than 20 s. Separated and simultaneously diluted blood plasma was directly injected into a capillary electrophoresis instrument with a contactless conductivity detector (CE-C4D) and analyzed in less than one minute. A separation electrolyte consisted of 10 mmol/L l-histidine, 15 mmol/L dl-glutamic acid, and 30 µmol/L cetyltrimethylammonium bromide. The whole procedure starting from the finger-prick sampling until the CE-C4D analysis was finished, took less than 5 min and was suitable for monitoring lactate increase in blood plasma during incremental cycling exercise. The observed lactate increase during the experiments measured by the developed CE-C4D method correlated well with the results from a hand-held lactate analyzer (R = 0.9882). The advantage of the developed CE method is the speed, significant savings per analysis, and the possibility to analyze other compounds from blood plasma.

Analysis of bicarbonate, phosphate and other anions in saliva by capillary electrophoresis with capacitively coupled contactless conductivity detection in diagnostics of gastroesophageal reflux disease.

Bicarbonate and phosphate constitute major salivary buffering components, and their importance consists in the neutralization of acidic gastric contents during reflux episodes. In this work, capillary electrophoresis with capacitively coupled contactless conductivity detector was applied for the analysis of bicarbonate, phosphate, and another inorganic (chloride, nitrite, nitrate, sulfate, thiocyanate) and organic anions (acetate, butyrate) to evaluate their levels in saliva. The background electrolytes of different composition and pH between 6.02-9.41 were assessed for the bicarbonate and phosphate determination by comparison of the real analyses of a model solution with the simulation by PeakMaster software. The optimized background electrolyte was composed of 10 mM 2-(N-morpholino)ethanesulfonic acid, 20 mM arginine, and 30 µM cetyltrimethylammonium bromide, pH 8.95. Using this BGE, the anion levels were compared in saliva from 20 patients suffering from gastroesophageal reflux disease (GERD) and saliva from 12 healthy subjects. Bicarbonate levels were significantly elevated in saliva from GERD patients suggesting the possible applicability of bicarbonate as a biomarker in non-invasive diagnostics of GERD by CE-C4 D.

Open source capillary electrophoresis.

Open source paradigm is becoming widely accepted in scientific communities and open source hardware is finding its steady place in chemistry research. In this review article, we provide the reader with the most up-to-date information on open source hardware and software resources enabling the construction and utilization of an "open source capillary electrophoresis instrument". While CE is still underused as a separation technique, it offers unique flexibility, low-cost, and high efficiency and is particularly suitable for open source instrumental development. We overview the major parts of CE instruments, such as high voltage power supplies, detectors, data acquisition systems, and CE software resources with emphasis on availability of the open source information on the web and in the scientific literature. This review is the first of its kind, revealing accessible blueprints of most parts from which a fully functional open source CE system can be built. By collecting the extensive information on open source capillary electrophoresis in this review article, the authors aim at facilitating the dissemination of knowledge on CE within and outside the scientific community, fosters innovation and inspire other researchers to improve the shared CE blueprints.

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples.

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

Evaluation of Important Analytical Parameters of the Peptest Immunoassay that Limit its Use in Diagnosing Gastroesophageal Reflux Disease.

GOAL: To evaluate the analytical parameters of a lateral flow (LF) pepsin immunoassay (Peptest) and assess its suitability in the diagnostics of gastroesophageal reflux disease (GERD). BACKGROUND: Peptest is a noninvasive assay to analyze pepsin in saliva, intended for use in GERD diagnostics. Although commercialized, fundamental studies on its performance are missing. The assay therefore requires basic analytical parameter evaluation to assess its suitability in clinical practice. STUDY: Assay reaction's time dependence, reader device repeatability, and individual LF devices and longitudinal pepsin concentration reproducibility in individual subjects was evaluated. Salivary pepsin was analyzed in 32 GERD patients with extraesophageal reflux symptoms and 13 healthy individuals. RESULTS: The assay's signal increase is not completed at the recommend readout time and continues to increase for another 25 minutes. The relative standard deviation of measurement was good when using the same LF device, ranging from 2.3% to 12.9%, but the reproducibility of 10 different individual LF devices was poor. The random error when analyzing the same saliva sample on 10 LF devices was as high as 36 ng/mL and this value is thus suggested as the positivity cut-off. Pepsin concentration in individual subjects during a 10-day period varied significantly. The sensitivity of the Peptest was 36.8% in the group with acid reflux and 23.1% in the group with weakly acid reflux. The specificity was 61.5%. CONCLUSIONS: The Peptest assay's sensitivity and specificity is low, the results are highly variable and it should not be used as a near-patient diagnostic method in primary care.

Capillary electrophoresis in the analysis of biologically important thiols.

In this review article, CE methods for analysis of biologically important thiols are overviewed. The article covers the period from the previously published comprehensive review in 2004 until mid-2016, with emphasis on various detection modes, novel approaches for sample preconcentration, and applications in clinical practice. The most commonly used detection methods, such as conductometry or absorbance detection, although universally applicable and available in most commercial instruments have low sensitivity and have only limited use in thiol analysis. Amperometric and MS detection are more sensitive and have their steady place in thiol analysis, although the mainstay remains CE with LIF detection, reaching nanomolar concentration sensitivities for most of the thiols. Novel probes for CE-LIF have been developed and tested. The preconcentration approaches using modified gold nanoparticles reaching excellent sensitivities in the picomolar range and various sample stacking methods are also reviewed. Finally, significant clinical applications of the developed methods are discussed with critical insights into the future of CE analysis of thiols.

New approach for cystic fibrosis diagnosis based on chloride/potassium ratio analyzed in non-invasively obtained skin-wipe sweat samples by capillary electrophoresis with contactless conductometric detection.

A new approach for sweat analysis used in cystic fibrosis (CF) diagnosis is proposed. It consists of a noninvasive skin-wipe sampling followed by analysis of target ions using capillary electrophoresis with contactless conductometric detection (C4D). The skin-wipe sampling consists of wiping a defined skin area with precleaned cotton swab moistened with 100 µL deionized water. The skin-wipe sample is then extracted for 3 min into 400 µL deionized water, and the extract is analyzed directly. The developed sampling method is cheap, simple, fast, and painless, and can replace the conventional pilocarpine-induced sweat chloride test commonly applied in CF diagnosis. The aqueous extract of the skin-wipe sample content is analyzed simultaneously by capillary electrophoresis with contactless conductometric detection using a double opposite end injection. A 20 mmol/L L-histidine/2-(N-morpholino)ethanesulfonic acid and 2 mmol/L 18-crown-6 at pH 6 electrolyte can separate all the major ions in less than 7 min. Skin-wipe sample extracts from 30 study participants-ten adult patients with CF (25-50 years old), ten pediatric patients with CF (1-15 years old), and ten healthy control individuals (1-18 years old)-were obtained and analyzed. From the analyzed ions in all samples, a significant difference between chloride and potassium concentrations was found in the CF patients and healthy controls. We propose the use of the Cl-/K+ ratio rather than the absolute Cl- concentration and a cutoff value of 4 in skin-wipe sample extracts as an alternative to the conventional sweat chloride analysis. The proposed Cl-/K+ ion ratio proved to be a more reliable indicator, is independent of the patient's age, and allows better differentiation between non-CF individuals and CF patients having intermediate values on the Cl- sweat test. Figure New approach for cystic fibrosis diagnosis based on skin-wipe sampling of forearm and analysis of ionic content (Cl-/K+ ratio) in skin-wipe extracts by capillary electrophoresis with contactless conductometric detection.

Capillary electrophoresis fingerprinting and spectrophotometric determination of antioxidant potential for classification of Mentha products.

In this work aqueous infusions from ten Mentha herbal samples (four different Mentha species and six hybrids of Mentha x piperita) and 20 different peppermint teas were screened by capillary electrophoresis with UV detection. The fingerprint separation was accomplished in a 25 mM borate background electrolyte with 10% methanol at pH 9.3. The total polyphenolic content in the extracts was determined spectrophotometrically at 765 nm by a Folin-Ciocalteu phenol assay. Total antioxidant activity was determined by scavenging of 2,2-diphenyl-1-picrylhydrazyl radical at 515 nm. The peak areas of 12 dominant peaks from CE analysis, present in all samples, and the value of total polyphenolic content and total antioxidant activity obtained by spectrophotometry was combined into a single data matrix and principal component analysis was applied. The obtained principal component analysis model resulted in distinct clusters of Mentha and peppermint tea samples distinguishing the samples according to their potential protective antioxidant effect. Principal component analysis, using a non-targeted approach with no need for compound identification, was found as a new promising tool for the screening of herbal tea products.

Single-breath analysis using a novel simple sampler and capillary electrophoresis with contactless conductometric detection.

The analysis of ionic content of exhaled breath condensate (EBC) from one single breath by CE with C(4) D is demonstrated for the first time. A miniature sampler made from a 2-mL syringe and an aluminum cooling cylinder for collection of EBC was developed. Various parameters of the sampler that influence its collection efficiency, repeatability, and effect of respiratory patterns were studied in detail. Efficient procedures for the cleanup of the miniature sampler were also developed and resulted in significant improvement of sampling repeatability. Analysis of EBC was performed by CE-C(4) D in a 60 mM MES/l-histidine BGE with 30 µM CTAB and 2 mM 18-crown-6 at pH 6 and excellent repeatability of migration times (RSD < 1.3% (n = 7)) and peak areas (RSD < 7% (n = 7)) of 12 inorganic anions, cations, and organic acids was obtained. It has been shown that the breathing pattern has a significant impact on the concentration of the analytes in the collected EBC. As the ventilatory pattern can be easily controlled during single exhalation, the developed collection system and method provides a highly reproducible and fast way of collecting EBC with applicability in point-of-care diagnostics.

Inorganic analysis using CE: advanced methodologies to face old challenges.

Inorganic species still remain of secondary importance when CE is used as an analytical separation tool. Despite considerable efforts of great many groups over last 20 years, plenty of competition, mostly from ion chromatography and elemental MS, restrains the routine use of CE in inorganic analysis. If there is yet hope clinging to make CE a viable alternative in the field, success would ultimately be attained through research activities on negating the disadvantages and amplifying the advantages of the method. Being the latest update of a series of reviews covering the time period from 1990 to 2010, this paper will comment on how and to what extent these challenges of inorganic CE analysis have been addressed in two recent years.

Fingerprinting postblast explosive residues by portable capillary electrophoresis with contactless conductivity detection.

A portable capillary electrophoretic system with contactless conductivity detection was used for fingerprint analysis of postblast explosive residues from commercial organic and improvised inorganic explosives on various surfaces (sand, concrete, metal witness plates). Simple extraction methods were developed for each of the surfaces for subsequent simultaneous capillary electrophoretic analysis of anions and cations. Dual-opposite end injection principle was used for fast (<4 min) separation of 10 common anions and cations from postblast residues using an optimized separation electrolyte composed of 20 mM MES, 20 mM l-histidine, 30 µM CTAB and 2 mM 18-crown-6. The concentrations of all ions obtained from the electropherograms were subjected to principal component analysis to classify the tested explosives on all tested surfaces, resulting in distinct cluster formations that could be used to verify (each) type of the explosive.

An open source 3D printed autosampler for capillary electrophoresis.

BACKGROUND: In-house built capillary electrophoresis (CE) systems represent a significant share of laboratory instrumentation. In most of these instruments, sample injection is effected manually with low to moderate precision and requires skilled operators. Although few automated samplers have been previously developed, typically only one sample at a time can be injected. If a series of samples is to be analyzed, manual intervention is required. In the present work, we developed and constructed a fully automated, open source, CE autosampler, able to handle up to 14 different samples that can be used as a modular component of any in-house built CE instrument. RESULTS: An inexpensive, 3D printed, open source, autosampler for CE was developed. The autosampler consists of two parts: an injection unit with carousel containing sample and electrolyte vials and a flushing unit, containing a miniature pressure/vacuum pump. The autosampler is operated by an Arduino Mega microcontroller and an Arduino code written in the laboratory. The injection sequence is entered through a keypad and LCD display by the user. The instrument can operate autonomously for extended periods of time. It was used for fully automated analysis and/or calibration of up to 14 samples with excellent injection repeatability reaching less than 2.7% RSD for peak areas. The sampler performance was tested with two independently built CE instruments, a CE system with contactless conductivity detection (C4D) and a CE system with laser induced fluorescence (LIF) detector. SIGNIFICANCE AND NOVELTY: A novel, 3D printed, Arduino-based autosampler for CE was developed. The autosampler allows autonomous hydrodynamic injection of up to 14 different samples with fully programmable injection sequence, including capillary flushing and high voltage and data acquisition control. It provides the missing instrumental sampling setup for laboratory made CE instruments. It can be simply constructed based on the open-source blueprints in any laboratory and be a useful and time-saving add-on to any modular CE instrument.

CE of inorganic species--a review of methodological advancements over 2009-2010.

This article is the seventh in a series examining biannually the methodological developments in the field of CE analysis of inorganic species and covers relevant documents published between January 2009 and December 2010. Following an analysis of the significant accomplishments that have impacted the field in two recent years, a survey of advances in general CE methodology is presented. Subsequently, several notable trends that can be perceived in this well-established field are discussed: the continuing rise of ME and consequent development of suitable detection techniques, most notably contactless conductivity detection, the constant pace of advances in speciation analysis, and an increase in non-analytical CE applications to study complexation and (bio)transformation reactions of metal analytes. A range of recently emerged multi-detection designs, ICP-MS interface devices, and separation systems, for which outpacing work has been conducted, are also brought into focus.

Determination of five priority haloacetic acids by capillary electrophoresis with contactless conductivity detection and solid phase extraction preconcentration.

A sensitive capillary electrophoretic separation method with contactless conductivity detection (C4D) for analysis of five priority haloacetic acids (HAA5) is presented. The analytes were baseline separated in an electrolyte composed of 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (HIS), and 30 µM cetyltrimethylammonium bromide (CTAB) at pH 6.0 in less than 4 min. A simplified solid-phase extraction (SPE) preconcentration procedure on highly cross-linked polystyrene-divinylbenzene (PS-DVB) type sorbent was developed and optimized with respect to short preconcentration time. HAA5 from a 25-mL sample aliquot of tap and swimming pool water could be preconcentrated in less than 5 min using an in-house made SPE column with recoveries ranging from 23 to 98%. Combining the SPE preconcentration procedure with capillary electrophoretic analysis, the attained limits of detection were between 6.1 and 12.2 µg/L with total analysis time of less than 10 min.

A novel temperature-controlled open source microcontroller based sampler for collection of exhaled breath condensate in point-of-care diagnostics.

Exhaled breath condensate (EBC) is an attractive, non-invasive sample for clinical diagnostics. During EBC collection, its composition is influenced by the collection temperature, a factor that is often not thoroughly monitored and controlled. In this study, we assembled a novel, simple, portable, and inexpensive device for EBC collection, able to maintain a stable temperature at any value between -7 °C and +12 °C. The temperature was controlled using a microcontroller and a thermoelectric cooler that was employed to cool the aluminum block holding the glass tube or the polypropylene syringe. The performance of the novel sampler was compared with the passively cooled RTube™ and a simple EBC sampler, in which the temperature was steadily increasing during sampling. The developed sampler was able to maintain a stable temperature within ±1 °C. To investigate the influence of different sampling temperatures (i.e., +12, -7, -80 °C) on the analyte content in EBC, inorganic ions and organic acids were analyzed by capillary electrophoresis with a capacitively coupled contactless conductivity detector. It was shown that the concentration of metabolites decreased significantly with decreasing temperature. The portability and the ability to keep a stable temperature during EBC sampling makes the developed sampler suitable for point-of-care diagnostics.

Quantification and identification of bile acids in saliva by liquid chromatography-mass spectrometry: Possible non-invasive diagnostics of Barrett's esophagus?

Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.