Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species.

Electrophoretic mobility shift assays are widely used in gel electrophoresis to study binding interactions between different molecular species loaded into the same well. However, shift assays can access only a subset of reaction possibilities that could be otherwise seen if separate bands of reagent species might instead be collisionally reacted. Here, we adapt gel electrophoresis by fabricating two or more wells in the same lane, loading these wells with different reagent species, and applying an electric field, thereby producing collisional reactions between propagating pulse-like bands of these species, which we image optically. For certain pairs of anionic and cationic dyes, propagating bands pass through each other unperturbed; yet, for other pairs, we observe complexing and precipitation reactions, indicating strong attractive interactions. We generalize this band-collision gel electrophoresis (BCGE) approach to other reaction types, including acid-base, ligand exchange, and redox, as well as to colloidal species in passivated large-pore gels.

Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10(-5) cm(2) V(-1) s(-1)) in the nanopores and in the nanochannel with the larger cross-sectional area.

Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research. Advances in techniques used for N-glycan structural identification include migration time indexing and exoglycosidase and lectin profiling, as well as mass spectrometry. Capillary electrophoresis methods have been developed that are capable of separating glycans with the same monosaccharide sequence but different positional isomers, as well as determining whether monosaccharides composing a glycan are alpha or beta linked. Significant applications of capillary electrophoresis to the analyses of N-glycans in biomarker discovery and biological therapeutics are emphasized with a brief discussion included on carbohydrate analyses of glycosaminoglycans and mono-, di-, and oligosaccharides relevant to food and plant products. Innovative, emerging techniques in the field are highlighted and the future direction of the technology is projected based on the significant contributions of capillary electrophoresis to glycoscience from 2014 to the present as discussed in this review.

Early stages of insulin aggregation, which involve the transient formation of oligomeric aggregates, are an important aspect in the progression of Type II diabetes and in the quality control of pharmaceutical insulin production. This study is the first to utilize capillary electrophoresis (CE) with ultraviolet (UV) detection to monitor insulin oligomer formation at pH 8.0 and physiological ionic strength. The lag time to formation of the first detected species in the aggregation process was evaluated by UV-CE and thioflavin T (ThT) binding for salt concentrations from 100 mM to 250 mM. UV-CE had a significantly shorter (5-8 h) lag time than ThT binding (15-19 h). In addition, the lag time to detection of the first aggregated species via UV-CE was unaffected by salt concentration, while a trend toward an increased lag time with increased salt concentration was observed with ThT binding. This result indicates that solution ionic strength impacts early stages of aggregation and β-sheet aggregate formation differently. To observe whether CE may be applied for the analysis of biological samples containing low insulin concentrations, the limit of detection using UV and laser induced fluorescence (LIF) detection modes was determined. The limit of detection using LIF-CE, 48.4 pM, was lower than the physiological insulin concentration, verifying the utility of this technique for monitoring biological samples. LIF-CE was subsequently used to analyze the time course for fluorescein isothiocyanate (FITC)-labeled insulin oligomer formation. This study is the first to report that the FITC label prevented incorporation of insulin into oligomers, cautioning against the use of this fluorescent label as a tag for following early stages of insulin aggregation.

Clinical analysis often requires rapid, automated, and high-throughput analytical systems. Microchip capillary electrophoresis (CE) has the potential to achieve very rapid analysis (typically seconds), easy integration of multiple analytical steps, and parallel operation. Although it is currently still in an early stage of development, there are already many reports in the literature describing the applications of microchip CE in clinical analysis. At the same time, more fully automated and higher throughput commercial instruments for microchip CE are becoming available and are expected to further enhance the development of applications of microchip CE in routine clinical testing. To put into perspective its potential, we briefly compare microchip CE with conventional CE and review developments in this technique that may be useful in diagnosis of major diseases.

Cystic fibrosis (CF) is the less rare and severe genetic disease among the European population. Biochemical diagnosis of CF is based on the demonstration of increased chloride concentration in sweat samples, obtained during the sweat test (ST). WynSep developed a capillary electrophoresis with contactless conductivity detection (CE-C4D) able to measure sweat chloride with a low sample volume. We evaluated the clinical feasibility of this device in a cohort of patients suspected of CF, in comparison with the common coulometric method (ChloroChek chloridometer).

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is "electrophoretically projected" into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm2 device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following in vitro synthesis.

In order to isolate an individual's genotype from a sample of biological material, most laboratories use PCR and Capillary Electrophoresis (CE) to construct a genetic profile based on polymorphic loci known as Short Tandem Repeats (STRs). The resulting profile consists of CE signal which contains information about the length and number of STR units amplified. For samples collected from the environment, interpretation of the signal can be challenging given that information regarding the quality and quantity of the DNA is often limited. The signal can be further compounded by the presence of noise and PCR artifacts such as stutter which can mask or mimic biological alleles. Because manual interpretation methods cannot comprehensively account for such nuances, it would be valuable to develop a signal model that can effectively characterize the various components of STR signal independent of a priori knowledge of the quantity or quality of DNA.

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

No abstract available

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.

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Given the endangered status of tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2-0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3-0.6), 1.2 (1-1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.

No abstract available

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE-MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE-MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE-MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE-MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE-MS analysis.

A technique to identify and quantitate simultaneously more than 30 compounds in individual neurons is described. The method uses nanoliter volume sampling, capillary electrophoresis separation, and wavelength-resolved native fluorescence detection. Limits of detection (LODs) range from the low attomole to the femtomole range, with 5-hydroxytryptamine (or serotonin [5-HT]) LODs being approximately 20 attomoles. Although the cellular sample matrix is chemically complex, the combination of electrophoretic migration time and fluorescence spectral information allows positive identification of aromatic monoamines, aromatic amino acids and peptides containing them, flavins, adenosine- and guanosine-nucleotide analogs, and other fluorescent compounds. Individual identified neurons from Aplysia californica and Pleurobranchaea californica are used to demonstrate the applicability and figures of merit of this technique.

This paper describes a target factor analysis based technique to model migration behavior in capillary electrophoresis (CE). The technique is effective in reducing the initial number of experiments required for modeling, as well as in recognizing the optimum conditions for running these experiments. The technique can be applied to optimize any number of parameters. The usefulness of the technique is demonstrated via both the experimentally obtained and the simulated data sets. Examples are provided to describe optimization of one and two parameters.

Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established.