Capillary Electrophoresis System Research Articles

Towards a radiation-tolerant contactless conductivity detector for use with capillary electrophoresis systems in spaceflight applications

The simplicity, versatility, and ease of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has led to its application for the analysis of a wide range of inorganic and organic species. While C4D detectors have relatively straightforward electronics, no designs specifically focused on spaceflight applications have been developed. The challenge lies in the limited set of components tolerant to high radiation levels and large temperature ranges when designing electronics for planetary science missions. In this work we present a C4D prototype designed for high radiation environments over the range of 50–300 krad total ionizing dose (TID). The proposed design, including selection of components, compatibility with radiation levels and the main differences compared to an open source version of the detector (OpenC4D) are discussed in detail. This prototype was also designed to allow monitoring of up to three capillaries for measurement redundancy. The Rad Hard C4D was assembled using COTS equivalents of the radiation-hardened counterparts, and its performance was evaluated by comparison to a commercial C4D device. Additionally, the reagents used for an astrobiology focused CE-C4D method developed previously were investigated for long-term storage and compatibility with high radiation levels. No loss in performance was observed for reagents exposed to 300 krad TID and stored for 2 years at room temperature.

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.

A handheld-type total integrated capillary electrophoresis system for SARS-CoV-2 diagnostics: Power, fluorescence detection, and data analysis by smartphone

A micro-capillary electrophoresis (μCE) system is one of the widely adopted techniques in the molecular diagnostics and DNA sequencing due to the benefits of high resolution, rapid analysis, and low reagent consumption, but due to the requirements of bulky high-power suppliers and an expensive laser-induced fluorescence detector module, the conventional set-up of μCE system is not adequate for point-of-care (POC) molecular diagnostics. In this study, we constructed a miniaturized and integrated μCE system which can be manipulated by a smartphone. The smartphone not only powers two boost converters and an excited laser, but also controls the relay for the power switch. Moreover, the complementary metal-oxide-semiconductor (CMOS) camera of the smartphone was used for detecting the fluorescence signal of amplicons amplified with reverse transcription-polymerase chain reaction (RT-PCR). We also developed a web-based application so that the raw data of the recorded fluorescence intensity versus the running time can display typical capillary electropherograms on the smartphone. The total size of the hand-held μCE system was 9.6 cm [Width] × 22 cm [Length] × 15.5 cm [Height], and the weight was ∼1 kg, which is suitable for POC DNA testing. In the integrated smartphone-associated μCE system, we could accurately analyze two genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely N gene and S gene along with two bracket ladders in 6 min to identify SARS-CoV-2. Such an advanced μCE platform can be applied for a variety of on-site molecular diagnostics fields with user-friendliness.

Capillary Nanogel Electrophoresis for the Determination of the β1-4 Galactosyltransferase Michaelis-Menten Constant and Real-Time Addition of Galactose Residues to N-Glycans and Glycoprotein.

A thermally reversible nanogel is used in capillary electrophoresis to create discrete regions for a galactosyltransferase reaction and separation. The β1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products, which were separated and identified. Using this capillary electrophoresis method, the degree of glycosylation was discernible for a pentasaccharide and for biantennary N-glycans. With the ability to distinguish between reaction products for which either one or two galactose residues were transferred, the capillary nanogel electrophoresis system was used to determine the Michaelis-Menten value, KM. For the β1-4 galactosyltransferase, the KM value obtained for a pentasaccharide substrate was 1.23 ± 0.08 mM. Once KM was established, the enzyme/substrate ratio was evaluated to add a single galactose residue or to fully galactosylate a biantennary N-glycan. Additionally, capillary nanogel electrophoresis was adapted to transfer galactose residues to protein. The applicability of the method for real-time online modification of whole protein was demonstrated with the Herceptin glycoprotein. Complete retardation by Erythrina cristagalli lectin after enzymatic modification confirmed the addition of galactose residues to the Herceptin. This demonstrated the potential of the method to be used for online modification of other glycoproteins.

Multimaterial 3D-printed contactless conductivity/laser-induced fluorescence dual-detection cell for capillary electrophoresis

利用多材料3D打印技术研制了用于毛细管电泳(CE)的二合一检测池,实现了电容耦合非接触电导(C4D)与共聚焦激光诱导荧光(LIF)两种检测方法在毛细管柱上同一位置同时检测。3D打印的检测池采用了导电的复合聚乳酸(PLA)材料制作C4D的屏蔽层,采用普通的绝缘PLA材料支撑C4D金属管电极并隔离屏蔽层。两根金属管电极通过“打印-暂停-打印”的方式嵌入到检测池中,两电极被2 mm厚的导电屏蔽层隔开,在屏蔽层中有一直径为1 mm的圆形通孔用于LIF检测。该检测池与带流通式进样接口的自组装CE系统联用,用于同时检测无机离子和异硫氰酸荧光素(FITC)标记的氨基酸。研究优化了C4D激励信号频率与电泳缓冲液浓度,选用的电泳缓冲溶液为10 mmol/L 3-吗啉丙烷-1-磺酸(MOPS)与10 mmol/L二(2-羟乙基)亚氨基三(羟甲基)甲烷(Bis-Tris)的混合溶液,选用C4D激励频率为77 kHz。二合一检测池应用于内径为25 μm的毛细管时,C 4D对Na+、K+和Li+的检出限分别为2.2、2.0和2.6 μmol/L; LIF对荧光素和FITC的检出限分别为7.6和1.7 nmol/L。两种检测方法的相对标准偏差在0.3%至4.5%之间(n=3),工作曲线的相关系数r 2≥0.9904。采用3D打印技术可以在实验室内实现复杂结构的制作,降低了制作的成本,且便于方法的推广和改进。

Evaluation of the trophic level and self-cleaning ability of small flows by hydrochemical indicators (through the example of the Rpen River)

Aim. Assessment by nitrification index values of the level of trophicity and toxicity for aquatic organisms in the waters of a small watercourse in an urbanized area by the content of nutrients in the water and its selfcleaning ability.Materials and Methods. The concentration of compounds of nutrients in the waters of the river was determined using the "Kapel 104T." system of capillary electrophoresis. Water was sampled for analysis according to GOST R 51592- 2000. The water content of readily oxidized organic compounds was assessed by the magnitude of permanganate oxidizability (PND F 14.1; 2.4.154-99) and difficultly oxidized compounds by the magnitude of the chemical oxygen consumption (RD 52.24.421-2012). Assessment of the water trophic level was evaluated by measuring the content of various forms of mineral nitrogen and mineral phosphorus. The aggregation index characterizing the toxicity of various forms of mineral nitrogen to aquatic organisms was calculated by their ratio to MPC for fishery reservoirs. The nitrifying ability of river waters was estimated by the value of the nitrification index (Initr.), calculated by the ratio of the concentration of nitrogen of nitrates in water to the sum of the concentrations of various forms of mineral nitrogen and the self-cleaning ability by the values of the nitrification index.Results. In terms of the aggregation index, water at all sampling points was found to be toxic. The river ecosystem is characterized by a weak ability to cleanse itself and a weak nitrifying ability, which is associated with the influence of numerous cyanotoxins on the microbiocenosis of the watercourse and filtration organisms.Conclusion. It was shown that the most sensitive indicator of trophicity of river waters is the concentration of the mineral phosphorus. Through measuring the ratio of the concentrations of mineral nitrogen to mineral phosphorus, it was established that blue-green algae (cyanobacteria), producers of cyanotoxins, dominate in the phytoplankton of the river in all the points studied.

Genetic diversity assessment and gene expression analysis of prolonged shelf-life genes in Mangalore melon (Cucumis melo ssp. agrestis var. acidulus)

Mangalore melon (Cucumis melo ssp. agrestis var. acidulus) is a non-dessert melon, extensively grown in the coastal districts of South India, but hardly known to the rest of the World. Immature or mature fruits of Mangalore melon are used in preparation of delicious dishes such as vegetable stew, chutneys and curries. They are appreciated for nutritional values, long shelf life and biotic stress resistance. Seventy-nine accessions of Mangalore melon were collected from five states of South India and their genetic diversity was assessed using inter simple sequence repeat (ISSR) markers. Putative candidate genes of extended shelf life in Mangalore melon were studied by quantitative reverse transcription polymerase chain reaction in comparison with cantaloupe (Cucumis melo L.). Shelf life varied from 65 to 300 days at room temperature. Six ISSR primers amplified 142 fragments ranging from 80 to 2380 bp with an average of 23.66 bands per marker on a high-resolution capillary electrophoresis system. Neighbor joining phylogenetic tree construction from the ISSR allele similarity based genetic distance revealed two major clusters with 46 and 33 accessions in each cluster. Expression of fruit ripening related genes of ethylene biosynthesis (1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase) and cell wall metabolism (polygalacturonase, xyloglucan endotransglucosylase/hydrolase and expansin) in Mangalore melons was significantly lower than the cantaloupe melon at 180 days after harvest. Mangalore melon is a promising genetic resource for enhancing the shelf life of melons. The putative candidate genes identified here are useful in enhancing shelf life of cantaloupe.

β-globin variants present in Western Sudan

Objective: A variety of observations of the hemoglobin D (Hgb D) phenotype has occurred in association with family studies of patients with sickle cell disease. Very little is known about the occurrence and prevalence of the Hgb D variant and its impact on blood profiles among Sudanese. This study was aimed at determining the percentage of Hb D in North Darfur State, West Sudan, whose population has been shown to have hemoglobin-S (Hb S) disease. Methods: From December 2017 to August 2018, this descriptive community-based investigation was conducted. Six hundred and sixty-six (666) people were randomly selected to participate in this study. With each participant's there was verbal consent. A questionnaire was designed to collect personal details. 5 mL of venous blood was gathered in EDTA containers. The Hb D variant was checked using Sebia Minicap Automated Capillary Electrophoresis System- USA and frequency was calculated using version 21.0 of the software package for social science (SPSS). Result: The prevalence of Hb variants was as follows: AD=0.6%, SS=2%, AS=10.5 %, and AA=86.9%. Conclusion: The prevalence of Hb D variant was 4 (0.6 %) in 666 participants from four western Sudanese tribes, beside reported of Hb SS and Hb AS.

The Unfolded Protein Response As a Predictor of Chemosensitivity in Pediatric Acute Myeloid Leukemia

Background: Survival in newly diagnosed pediatric patients with acute myeloid leukemia (AML) is approximately 65% with a much lower survival in patients with relapsed disease. Leukemia cells are exposed to proteotoxic stress due to their rapid, inefficient metabolism and accumulation of reactive oxygen species. This proteotoxic stress results in the production of misfolded proteins, which leads to activation of cell stress pathways, such as the unfolded protein response (UPR). We hypothesized that UPR induction correlates with a better response to chemotherapy.Methods: Peripheral blood samples from pediatric patients with AML were collected prior to initiation of induction chemotherapy, then at ~6 hours (h) and 24 h post initiation of chemotherapy. Peripheral blood mononuclear cells were frozen as pellets after depletion of non-tumor cells using magnetic bead selection (Miltenyi Biotech). The expression of UPR proteins was determined by chemiluminescence using an automated capillary electrophoresis system, ProteinSimple WesTM. The expressions of 5 UPR proteins were examined including Grp78, IRE1, total and phosphorylated (active) eIF2-α, and ATF6-α. Protein expression was normalized to actin expression and fold differences were compared between samples and within each sample at different time points.Results: Within each group of UPR proteins, there was a wide variability in the level of baseline expression, with 6 of 38 samples expressing >4 fold increase in Grp78, compared to the median level in healthy pediatric and adult controls (Figure 1). Basal IRE1 levels were also significantly higher in pediatric controls vs. adult controls. A trend towards lower overall survival (OS) and event-free survival (EFS) was noted in patients who had a low baseline Grp78 and a low baseline IRE1 (low baseline IRE1 was defined as <2 x below the median level of healthy pediatric controls). Post chemotherapy, UPR induction (defined as >2 x above baseline level) was seen in a subset of patients, most notably for ATF6α. However, post chemotherapy UPR induction was not associated with differences in either event-free survival or overall survival.Conclusion: UPR has a variable expression at baseline in pediatric AML patients, with a trend for lower overall survival in patients with a low basal Grp78 and IRE1 expression, suggesting chemoresistance in this subset of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of a Capillary Electrophoresis System for the Separation of Proteins.

Serum protein electrophoresis is one of the core investigations for screening for monoclonal proteins. Among the available capillary systems, the Helena V8 system has been evaluated in a limited number of studies. In total, 310 sera samples were assessed on the Helena V8 system and compared with the Sebia Capillarys instrument. Abnormalities suggestive of monoclonal proteins were confirmed by immunofixation. Imprecision studies and reference intervals were determined. The imprecision of the Helena V8 was inferior or equal to 5.8%. The mean bias of Helena V8 vs Sebia Capillarys was about -0.9 g/L for albumin; -0.2 g/L for alpha-1; 1.1 g/L for alpha-2; -0.2 g/L for beta; 0.3 g/L for gamma; -0.5 g/L for monoclonal protein in beta; and 0.3 g/L for monoclonal protein in gamma. Among the 56 samples with monoclonal proteins confirmed by immunofixation, all were seen on both methods, with only 1 discordant result at a cutoff of 5.0 g/L. Reference intervals were statistically different between the 2 analyzers, except for the beta fraction. Our evaluation confirms the good analytical performance of the Helena V8 analyzer as a suitable alternative to the Sebia Capillarys instrument.

INFLUENCE OF PHYTOLYTIC AND PROTEOLYTIC ENZYMES ON CONVERSION OF WHEAT AND CORN GRAIN POLYMERS

Grain, in addition to starch, hemicelluloses and protein, is known to be phytic acid and its salts. It has been shown that the use of phytolytic enzymes promotes the release of phosphorus, improves the digestibility of feed nutrients and increases the meat productivity of livestock and poultry. However, the catalytic effect of phytases and their combinations with proteases on degradation of polysaccharides and protein polymers in grain raw materials, on the release of cations and anions, especially in grain wort for the production of alcohol, has been little studied. This work shows for the first time that phytolytic enzymes improve qualitative estimates, rheological properties and change the ionic composition of grain wort by not only releasing phosphates and metal cations, but also by increasing the concentration of organic salt anions. The combined action of phytolytic and proteolytic enzymes on polymers of grain raw materials provides their catalytic degradation to soluble carbohydrates, nitrogenous substances, cations and anions to produce the enriched grain wort. This work aimed to assess the degree of phytolytic and proteolytic conversion of high-molecular-weight polymers in grain wort. For grain wort, 50 g portions of wheat (Triticum sp.) or corn (Zea mays) grain flour put into Erlenmeyer flasks were added with 150 cm3 of water and incubated in a water bath. At grain batching for starch dextrinization, a thermo-stable a-amylase was added (0.6 units/g starch). For starch conversion to sugars and hydrolysis of non-starch polysaccharides in the control, we used glucoamylase (9.0 units/g starch) and xylanase (0.15 units/g raw material). Phytase (from 1.0 to 2.5 units/g raw material) and a proteolytic enzyme preparation (0.1 units/g raw material) were also added. The control was added neither with phytase, nor the proteolytic enzyme. The profiles of the main polymers of grain raw materials, the grain wort concentration, the content of reducing carbohydrates (RС) were determined according to the techno-chemical instructions for the control of alcohol production, the amine nitrogen (NH2+) concentration was measured by a method based on the ability of amino acids to form soluble copper compounds with a suspension of copper phosphate. The dynamic viscosity of the grain wort was evaluated by vibrational viscometry. The study of the ionic composition of the grain mix and wort was carried out using a PrinCE-560 series capillary electrophoresis system (PrinCE Technologies B.V., Netherlands) equipped with a conductometric detector. The optimal dosage of phytase was 1.5 units/g raw material which ensures the maximum release of ions identified by capillary electrophoresis and the effective conversion of polysaccharides, protein and phytin substances of the grain. It was found that the phytolytic enzymes contributed to a decrease in the viscosity of wheat and corn wort of more than 20 %, a 9.5-11.3 % increase in the concentration of reducing carbohydrates, and a 2.1-2.4-fold increase in the concentrations of released ions. The concentration of amino nitrogen in the wort did not change significantly. It was shown that as a result of the phytolytic action, the concentration of not only metal phosphates and cations, but also anions of organic salts, such as oxalates, malates, citrates, and succinates (in wheat wort) and oxalates, malates, citrates, and lactates (in corn wort) increased. A more significant effect of phytase for corn wort was revealed: the concentration of phosphates in the nutrient medium increased 3.9 times vs. 1.6 times for wheat wort, the levels of potassium and magnesium ions were 12 % and 22 % higher, respectively, as compared to the control in which the phytase was not used. The optimal composition of the enzyme complex is proposed which ensures the effective hydrolysis of polysaccharides, phytin and protein substances of processed raw materials. The synergistic effect of phytolytic and proteolytic enzymes enhances the catalytic hydrolysis of high molecular weight polymers of plant origin and enriches the grain wort with carbohydrates, nitrogenous substances, phosphates, and minerals in bioavailable form. Biocatalytic treatment of grain with the developed enzyme complex which, along with amylases and xylanase, contains phytase and proteases, provides a 16.8 % and 18.8 % increase in the concentration of reducing carbohydrates in wheat and corn wort, a 1.7- and 1.9-fold increase in amine nitrogen and a decrease in wort viscosity by 41.7 % and 44.7 %, respectively.

КОНВЕРСИЯ ПОЛИМЕРОВ ЗЕРНА ПШЕНИЦЫ И КУКУРУЗЫ ПОД ВЛИЯНИЕМ ФИТОЛИТИЧЕСКИХ И ПРОТЕОЛИТИЧЕСКИХ ФЕРМЕНТОВ

Grain, in addition to starch, hemicelluloses and protein, is known to be phytic acid and its salts. It has been shown that the use of phytolytic enzymes promotes the release of phosphorus, improves the digestibility of feed nutrients and increases the meat productivity of livestock and poultry. However, the catalytic effect of phytases and their combinations with proteases on degradation of polysaccharides and protein polymers in grain raw materials, on the release of cations and anions, especially in grain wort for the production of alcohol, has been little studied. This work shows for the first time that phytolytic enzymes improve qualitative estimates, rheological properties and change the ionic composition of grain wort by not only releasing phosphates and metal cations, but also by increasing the concentration of organic salt anions. The combined action of phytolytic and proteolytic enzymes on polymers of grain raw materials provides their catalytic degradation to soluble carbohydrates, nitrogenous substances, cations and anions to produce the enriched grain wort. This work aimed to assess the degree of phytolytic and proteolytic conversion of high-molecular-weight polymers in grain wort. For grain wort, 50 g portions of wheat (Triticum sp.) or corn (Zea mays) grain flour put into Erlenmeyer flasks were added with 150 cm3 of water and incubated in a water bath. At grain batching for starch dextrinization, a thermo-stable a-amylase was added (0.6 units/g starch). For starch conversion to sugars and hydrolysis of non-starch polysaccharides in the control, we used glucoamylase (9.0 units/g starch) and xylanase (0.15 units/g raw material). Phytase (from 1.0 to 2.5 units/g raw material) and a proteolytic enzyme preparation (0.1 units/g raw material) were also added. The control was added neither with phytase, nor the proteolytic enzyme. The profiles of the main polymers of grain raw materials, the grain wort concentration, the content of reducing carbohydrates (RС) were determined according to the techno-chemical instructions for the control of alcohol production, the amine nitrogen (NH2+) concentration was measured by a method based on the ability of amino acids to form soluble copper compounds with a suspension of copper phosphate. The dynamic viscosity of the grain wort was evaluated by vibrational viscometry. The study of the ionic composition of the grain mix and wort was carried out using a PrinCE-560 series capillary electrophoresis system (PrinCE Technologies B.V., Netherlands) equipped with a conductometric detector. The optimal dosage of phytase was 1.5 units/g raw material which ensures the maximum release of ions identified by capillary electrophoresis and the effective conversion of polysaccharides, protein and phytin substances of the grain. It was found that the phytolytic enzymes contributed to a decrease in the viscosity of wheat and corn wort of more than 20 %, a 9.5-11.3 % increase in the concentration of reducing carbohydrates, and a 2.1-2.4-fold increase in the concentrations of released ions. The concentration of amino nitrogen in the wort did not change significantly. It was shown that as a result of the phytolytic action, the concentration of not only metal phosphates and cations, but also anions of organic salts, such as oxalates, malates, citrates, and succinates (in wheat wort) and oxalates, malates, citrates, and lactates (in corn wort) increased. A more significant effect of phytase for corn wort was revealed: the concentration of phosphates in the nutrient medium increased 3.9 times vs. 1.6 times for wheat wort, the levels of potassium and magnesium ions were 12 % and 22 % higher, respectively, as compared to the control in which the phytase was not used. The optimal composition of the enzyme complex is proposed which ensures the effective hydrolysis of polysaccharides, phytin and protein substances of processed raw materials. The synergistic effect of phytolytic and proteolytic enzymes enhances the catalytic hydrolysis of high molecular weight polymers of plant origin and enriches the grain wort with carbohydrates, nitrogenous substances, phosphates, and minerals in bioavailable form. Biocatalytic treatment of grain with the developed enzyme complex which, along with amylases and xylanase, contains phytase and proteases, provides a 16.8 % and 18.8 % increase in the concentration of reducing carbohydrates in wheat and corn wort, a 1.7- and 1.9-fold increase in amine nitrogen and a decrease in wort viscosity by 41.7 % and 44.7 %, respectively.

A Comparison of gel (Hydragel 30) and capillary (Capillarys III Tera) electrophoresis for the characterization of human serum proteins

ObjectivesTo compare gel (Hydrasys 2 from Sebia) and capillary (Capillarys III Tera, Sebia) electrophoresis for the characterization of human serum proteins. Design and methods304 sera tested by gel electrophoresis during 8 routine laboratory days were concurrently tested by capillary electrophoresis. Gels were read by an experienced medical technologist while capillary profiles by a Sebia representative and the same technologist. Most sera (214 of 304, 70%) were also analyzed by immunofixation electrophoresis, used here as the gold standard to calculate sensitivity and specificity of the gel and capillary systems. ResultsGel and capillary estimated the concentration of albumin, gamma region, and M-spikes nearly perfectly, and that of beta, alpha-2, and alpha-1 regions with excellent correlation. The two systems classified concordantly 268 of 304 sera (88% agreement) as having no, one, or two M-spikes, but differed in the remaining 36 sera (12%). Gel electrophoresis correctly identified M-spikes in 82 of 112 sera that were shown to have monoclonal band(s) by immunofixation (73% sensitivity), and correctly did not reveal M-spikes in 97 of the 102 sera that had no immunofixation bands (95% specificity). Capillary achieved slightly higher sensitivity (85 of 112, 76%) and slightly lower specificity (94 of 102, 92%), but the two areas under the ROC curves were nearly identical at 0.84. ConclusionsGel and capillary electrophoresis systems perform similarly to estimate the concentration of serum protein fractions and detect M-spikes.

Investigation of Binding behaviour of a water-soluble gallium (III) phthalocyanine with double-stranded and G-quadruplex DNA via experimental and computational methods

DNA is an important target for the development of chemotherapeutic drug design due to increased and uncontrolled cell proliferation of cancer cells. In the present study, the affinity of water-soluble gallium(III) phthalocyanine chloride (GaPc) non-peripherally substituted with 2-mercapto-N-methylpyridinium groups to different types of DNA (double-stranded ctDNA and G-quadruplex DNA) was investigated. The interaction mechanisms were evaluated by UV-Vis, fluorescence and circular dichroism spectroscopic techniques, competitive dialysis assay, and capillary gel electrophoresis (CGE) system integrated with a high sensitivity LED-induced fluorescence (LIF) detector. Binding mode studies were carried out by molecular docking and molecular dynamic simulations. The experimental binding constants were determined as 1.9 × 106 M−1, 1.1 × 106 M−1, 8.5 × 105 M−1, 2.9 × 104 M−1 for c-MYC, AS1411, Tel21, and ctDNA, respectively. Competitive binding assay confirmed the higher affinity towards G-quadruplex structure rather than ctDNA. The GaPc-G-quadruplex DNA conjugates showed higher Kb constants and a strong absorption band closer to NIR window which is a desired feature for the photodynamic therapy applications. Besides, experimental data show that the formation of GaPc-G-quadruplex DNA conjugates reduced the aggregation of GaPc. Reduction of aggregation of phthalocyanine is important. Because aggregation decreases the photoactivity of most photosensitizers. The experimental results suggested that both intercalative and non‑intercalative binding contributions occurred in the case of double-stranded ctDNA. Confirming experiments, molecular docking studies showed that the GaPc can bind from two different major groove regions in double-stranded DNA while only one binding mode was observed in the G-quadruplex DNA. Both experimental and computational studies suggested that the conformational change and unfolding mechanism in G-quadruplex DNA structure were determined upon interaction with GaPc.

Simultaneous separation and determination of three huperzine alkaloids in Huperzia serrata and its preparations by cyclodextrin-modified mixed micellar electrokinetic capillary chromatography

In this study, a simple and sensitive cyclodextrin-modified mixed micellar electrokinetic capillary chromatography (CD-MEKC) method has been developed for the simultaneous separation and determination of Huperzine A (HupA), Huperzine B (HupB) and Huperzine C (HupC) in Huperzia serrata (H. serrata). The optimal conditions (pH 9.3) were composed of 10 mM sodium tetraborate solution, 40 mM sodium dodecyl sulfate (SDS), 50 mM sodium cholate (SC) and 3.0 mM mono-(6-ethylenediamine-6-deoxy)-β-cyclodextrin (ED-β-CD). The separation and determination process were performed on a P/ACE MDQ capillary electrophoresis system, the separation voltage was 15 kV, the temperature was 25 °C and the detection wavelength was 308 nm. Under the optimum conditions, the migration time was less than 9 min. The LOD and LOQ were between 0.38 and 0.80 μg/mL and 1.2–2.3 μg/mL, respectively. The developed method, with excellent precision and accuracy, was applied for the determination of three alkaloids in H. serrata and its formulations.

From environmental bioavailability of metal(loid)s to their ecogenotoxicological effects in land snails.

To date, no study has linked the environmental and the ecogenotoxicological bioavailability of contaminants to land snails. Yet, understanding the specific ecotoxicological mechanisms from bioaccumulation to genotoxicity is necessary e.g., to build an adverse outcome pathway relevant to risk assessment. Consequently, the aim of our study is to look for relationships between accumulated concentrations of As, Cd and Hg in sub-adult snails and ecotoxicological effects at the individual (survival and growth) and molecular (genomic stability) levels. This study combines random amplified polymorphic DNA (RAPD) coupled with high-resolution capillary electrophoresis system (HRS) and micronucleus (MN) assay on haemocytes to consider various types of cytogenomic damage, such as chromosomal aberrations, breakages, adducts and mutations. The results showed alteration of the individual endpoints at higher accumulation quotients (AQs) that reflect the excess of transfers to snails, especially with decreased survival for As. In addition, genotoxic effects were observed with an increased occurrence of MN in haemocytes for the three meta(loid)s considered (R2 from 0.57 to 0.61 as a function of the meta(loid)s). No concentration-dependent decrease in genome stability was highlighted by RAPD-HRS in snails exposed to As and Cd but not Hg. Our results demonstrate the complementarity of the RAPD-HRS and the MN assay for understanding the different genotoxic mechanisms of the three metal(loid)s studied in land snails. They show a way to better assess environmental risks of contaminated soils by associating ecotoxicity, genotoxicity and bioaccumulation assays (ISO 24032), i.e., ecogenotoxicological bioavailability. Convergences highlighted here between the bioaccumulation of metal(loid)s in viscera and genotoxic effects in haemocytes constitute a way to better assess the bioavailability of contaminants in soils to the land snail and the subsequent environmental risk.

COMPREHENSIVE EVALUATION OF SOYBEAN AND SUNFLOWER PROCESSING PRODUCTS

The literature review of the article presents the characteristics of the protein value of plant feed materials, in particular, of soybean and sunflower processing products, depending on the production technology. Their amino acid composition ideally meets the needs of young animals and poultry due to the high content of essential amino acids.&#x0D; The section «Materials and methods» presents methods for determining: crude protein by the Kjeldahl method, soluble protein in 0.2% potassium hydroxide, Barnstein protein, falsification of protein products with inorganic nitrogen, as well as characteristics of methods for determining the content of essential and non-essential amino acids, incl. sulfur-containing, using the system of capillary electrophoresis «Drops-105M».&#x0D; As a result of the research it was found that of the total number of analyzed samples of soybean meal in terms of protein content, 30.8% of products did not meet the declared values of producers and were in the range of 37.4 - 40.8%, in terms of protein solubility were in the range of 75 - 78%, which meets the norms of feeding.In addition, 5.0% of the total number of samples analyzed, were falsified after Bard alcohol, falsification of inorganic nitrogen was not found. The use of low-protein raw materials for the production of sunflower meal and cake was reflected in their protein content and ranged from 26.3 to 33.7%.&#x0D; Fermented soybean product and toasted soybean meal are characterized by a higher total content of essential amino acids in the protein, which was 26.3% and 27.2%, respectively, compared with full-fat expanded soybean expanded, where this figure was at 20.25%, and prevailed other analyzed products by methionine, threonine, tyrosine, phenylalanine, proline, alanine etc.&#x0D; The analyzed sample of sunflower meal, produced by a special technology is equivalent in total amino acid content in the studied soy products. Its content of methionine was 1.5 and 2.1 times higher, compared with toasted soybean meal and full-fat soybean, respectively. Also, the content of threonine, glycine, cystine, tryptophan, aspartic and glutamic acids in the sample of sunflower meal prevailed the content of the same amino acids in soybean products.&#x0D; Thus, the comprehensive assessment of plant protein components makes it possible to control not only the quantitative content of protein and amino acids, but also provides a detailed assessment of feeding.

Single-Run Separation and Quantification of 14 Cannabinoids Using Capillary Electrophoresis

Quantification of major cannabinoids in cannabis products is normally performed using high-pressure liquid chromatography (HPLC)-based methods. We propose a cost-effective alternative method that successfully separates and quantifies 14 cannabinoids in a single run using capillary electrophoresis (CE) coupled with a UV detector in 18 min. The separation is carried out in 60% acetonitrile in the presence of 6.5 mM sodium hydroxide and 25 µM β-cyclodextrin, resulting in good separation of cannabinoids. Our CE method demonstrated the limit of detection between 1.2–1.8 µg/mL, with the linear range reaching up to 50 µg/mL. We validated the method performance by testing a plant extract and quantifying cannabinoid content. This method is the first to separate 14 cannabinoids in one run using a CE system with UV detection.

Rapid Analysis for Staphylococcus aureus via Microchip Capillary Electrophoresis.

Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureus nuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/μL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.

High-resolution Arduino-based data acquisition devices for microscale separation systems

In this work, we have designed, constructed, and evaluated simple, inexpensive open-source data acquisition systems based on various analog-to-digital converter modules (ADS 1115, MCP 3424, LTC 2400, with resolution from 16 to 24-bit) and a miniature Arduino Nano ™ microcontroller. The constructed data acquisition systems provide excellent performance and are comparable to a commercial, 24-bit device. We provide full schematics and corresponding source codes so that analytical chemists can easily construct any of the developed systems without extensive electronic or programming knowledge. The 24-bit LTC 2400 based device provided the best and comparable performance to a commercial, high-end 24-bit sigma to delta converter (ORCA 2800) at a fraction of cost (less than 50 USD compared to 870 USD for the commercial counterpart). The excellent performance was verified using a capillary electrophoresis system with contactless conductivity detection and separation of inorganic ions in clinical skin wipe and tap water samples.