Capillary Electrophoresis Detection Research Articles

Electric field-enhanced backscatter interferometry detection for capillary electrophoresis

Backscatter interferometry (BSI) is a refractive index (RI) detection method that is easily integrated with capillary electrophoresis (CE) and is capable of detecting species ranging from inorganic ions to proteins without additional labels or contrast agents. The BSI signal changes linearly with the square of the separation voltage which has been used to quantify sample injection, but has not been explored as a potential signal enhancement mechanism in CE. Here we develop a mathematical model that predicts a signal enhancement at high field strengths, where the BSI signal is dominated by the voltage dependent mechanism. This is confirmed in both simulation and experiment, which show that the analyte peak area grows linearly with separation voltage at high field strengths. This effect can be exploited by adjusting the background electrolyte (BGE) to increase the conductivity difference between the BGE and analyte zones, which is shown to improve BSI performance. We also show that this approach has utility in small bore capillaries where larger separation fields can be applied before excess Joule heating degrades the separation. Unlike other optical detection methods that generally degrade as the optical pathlength is reduced, the BSI signal-to-noise can improve in small bore capillaries as the larger separation fields enhance the signal.

Early screening of thalassemia in pregnant women in northern China by capillary electrophoresis for the determination of hemoglobin electrophoresis.

The objective of this study was to analyze the effectiveness of capillary electrophoresis detection of hemoglobin electrophoresis (HE) for the early screening of thalassemia. In the first choice, 974 pregnant women were selected for capillary electrophoresis to detect HE, which showed that 46 of them were abnormal (4.72%), including 16 cases with HbA2<2.5% and 28 cases with HbA2>3.5% and/or HbF≥2.0%. In one case each of HbH and HbBart's abnormal bands was found. The genotype test results showed the presence of thalassemia in 34 cases, using the genotype test results as the gold standard, after calculation it was seen that capillary electrophoresis for HE diagnosis of the occurrence of thalassemia had a sensitivity and specificity of 54.34% and 70.97% (P<0.05). These results suggest that in the screening of thalassemia in northern China, capillary electrophoresis for HE has good application and can be used as one of the routine screening tools, but further confirmation by genotype testing is still needed.

Compact contactless conductometric, ultraviolet photometric and dual-detection cells for capillary electrophoresis via additive manufacturing

The growing demand for tailored detectors in capillary electrophoresis (CE), addressing tasks like field deployment or dual-detection analysis, emphasizes the necessity for compact detection cells. In this work, we propose cost-effective and user-friendly additive manufacturing (3D-printing) approaches to produce such miniaturized detection cells suitable for a range of CE applications. Firstly, capacitively-coupled contactless conductivity detection (C4D) cells of different sizes are fabricated by casting low-melting-point alloy into 3D-printed molds. Various designs of Faraday shields are integrated within the cells and compared. A mini-C4D cell (9.5×7.0×7.5 mm3) is produced, with limits of detection for alkaline cations ranging from 8-12 μM in a short–capillary based CE application. Secondly, ultraviolet photometric (UV-PD) detection cells are fabricated using 3D printing. These cells feature two narrow slits with a width of 60 μm, which are positioned along the path of incident and transmission light to facilitate collimation. A deep UV-LED (235 nm or 255 nm) is employed as the light source, and black resin is determined to be the optimal material for 3D printing the UV-PD cell, owing to its superior UV light absorption capabilities. The UV-PD cell is connected to the LED and photodetector through two optical fibers, making it easy to switch the light source and detector. The effective pathlength and stray light percentage for detecting on a 75 μm id capillary are 74 μm and 0.5 %, respectively. Thirdly, a dual-detection cell that combined C4D and UV-PD at a single detection point is proposed. The performance of direct detection by C4D and indirect detection by UV-PD is compared for detecting organic acids. The strategies for developing cost-effective compact detection cells facilitate the versatile integration of multiple detection methods in CE analysis.

Capillary electrophoresis on-line hyphenated with mass spectrometry for analysis of insulin-like growth factor-1 in pharmaceutical preparations.

Insulin-like growth factor-1 (IGF-1) is a 70-amino acid single-chain polypeptide, which has found application in diagnostics as a biomarker of growth hormone disorders and as a therapy for growth failure in children and adolescents. Due to its strong anabolic effects, it is often abused by athletes for doping purposes. Here, we developed an on-line hyphenated method based on capillary zone electrophoresis (CZE) and triple quadrupole mass spectrometry (MS) detection with electrospray ionization (CZE-electrospray ionization source-MS [CZE-ESI-MS]) for the determination of IGF-1 in pharmaceutical matrices. We achieved a highly efficient, accurate, repeatable, sensitive, and selective analysis of IGF-1 with favorable migration times (<15min). Optimized and validated CZE-ESI-MS method was successfully applied for the determination of IGF-1 in injectable solutions (Increlex®), and its presence was also confirmed in nutritional preparations (tablets and liquid colostrum). This is the first validated CZE-ESI-MS method for the determination of IGF-1 in pharmaceutical matrices revealing the potential of capillary electrophoresis for its use in drug quality control laboratories with benefits, such as high separation efficiency, high-speed analysis, low sample consumption, as well as environmental and cost aspects.

Capillary surface modification using millimolar levels of aminosilane reagent for highly efficient separation of phenolic acids and flavonols by capillary electrophoresis with UV detection.

Phytochemical analysis of phenolic acids and flavonols poses a challenge, necessitating the development of an efficient separation method. This facilitates the quantification of these compounds, yielding valuable insights into their benefits. To develop a highly effective separation of phenolic acids and flavonols by capillary electrophoresis and ultraviolet (UV) detection through the modification of the capillary surface using 3-aminopropyltriethoxysilane (APTES) at millimolar concentrations. The capillary surface is modified with 0.36 mM-APTES solution. The electrolyte is 20.0 mM borate buffer (pH9.0). Separation performance (plate number N, resolution Rs ), stability, and reproducibility of the coating procedure are evaluated using the analysis of phenolic acids, rutin and quercetin. The modified capillary provided efficient separation with plate numbers N ≥ 1.0 × 104 m-1 and resolution Rs ≥ 0.8 for all pairs of adjacent peaks of the separation of five selected phenolic acids, rutin, quercetin, caffeine and methylparaben (as internal standard). The precisions of the relative migration times for 17 consecutive analyses of samples over 3h were 1% relative standard deviation (RSD) for rutin and 7% RSD for quercetin. The analysis of rutin and quercetin in 12 dietary supplement product samples only required a simple dilution step for sample preparation. A straightforward modification technique utilising millimolar concentrations of APTES resulted in highly efficient separation of phenolic acids, rutin and quercetin, accompanied by high precision and surface stability. The modified capillary proved successful in analysing rutin and quercetin content in dietary supplements.

Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer

Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Sensitive determination of bisphenols in environmental samples by magnetic porous carbon solid-phase extraction combined with capillary electrophoresis

Bisphenol compounds exist widely in the environment and pose potential hazards to the environment and human health, which has aroused widespread concern. Therefore, there is an urgent need for an efficient and sensitive analytical method to enrich and determine trace bisphenols in environmental samples. In this work, magnetic porous carbon (MPC) was synthesized by one-step pyrolysis combined with a solvothermal method for magnetic solid-phase extraction of bisphenols. The structural properties of MPC were characterized by field emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and saturation magnetization analysis. Its adsorption properties were evaluated by adsorption kinetics and adsorption isotherm studies. By optimizing the magnetic solid-phase extraction and capillary electrophoresis separation conditions, a capillary electrophoresis separation and detection method for four bisphenols was successfully constructed. The results showed that the detection limits of the proposed method for the four bisphenols were 0.71–1.65 ng/mL, the intra-day and inter-day precisions were 2.27–4.03% and 2.93–4.42%, respectively, and the recoveries were 87.68%-108.0%. In addition, the MPC could be easily recycled and utilized, and even if the magnetic solid-phase extraction was repeated 5 times, the extraction efficiency could still be kept above 75%.

The development of the novel 22 X- indel multiplex system for forensic genetics

InDel (Insertions/deletion) markers have been used as an alternative, or as a complementary marker system, to STR markers in human identification due to their advantages such as low mutation rates, no stutter, and potential small amplicon sizes. In forensic sciences, sex chromosomes are widely used in forensic genetics for specific cases. For example, the relationship between father and daughter can be determined by using X-InDels. In this study, we developed a novel 22 X-InDel multiplex system that was identified by two separate assays with fluorescence amplification and capillary electrophoresis detection technology. We chose 22 X-InDel markers based on the following criteria: mean heterozygosity over 30% in Europeans; minimum of 250 Kb differences between each InDel loci; and an amplicon length that was less than 300 bp. We performed an optimization and validation study of 22 X-InDel systems under the following parameters: analytical threshold, sensitivity, precision and accuracy, stochastic threshold, repeatability, and reproducibility. We evaluated the allele frequency of this multiplex system in the Turkish population, and then the population comparisons were carried out on data from 1000 Genome populations (Europe, Africa, America, South Asia, and East Asia). The sensitivity test showed a complete genotyping profile with DNA concentrations as low as 0.5 ng. The heterozygosity ratio of 22 X-InDel loci was determined as 0.4690 and the discrimination power was defined as 0.99. The results show that the new 22 X-InDel multiplex system provides high polymorphism information, and it is a reproducible, accurate, sensitive, and robust system that could be used as an additional tool for kinship testing.

Highly Sensitive In-Capillary Derivatization and Field Amplified Sample Stacking to Analyze Narcotic Drugs in Human Serum by Capillary Zone Electrophoresis

An in-capillary derivatization (in-CAP-D) integrated with field amplified sample stacking (FASS) has been developed for the determination of morphine (MOR) and its metabolite, morphine-6-glucuronide (MOR-6-G) in human serum using capillary zone electrophoresis (CZE) and fluorescence detection (in-Cap-D-FASS-CZE). Acetonitrile was employed in removing proteins and extracting MOR and MOR-6-G into the clear supernatant containing codeine (COD) as an internal standard (IS). The derivatization was achieved in an in-capillary mode by introducing the acetonitrile-treated samples into a running electrolyte containing an oxidizing agent of potassium ferricyanide, whereas MOR, MOR-6-G and COD were oxidized into dimer derivatives with highly fluorescent intensity. The effectiveness and sensitivity of the in-Cap-D-FASS-CZE method were affected by many parameters, and the following conditions were found to be optimal: 70 m Mdisodium tetraboratedecahydrate (pH, 10.5), 0.30 mM ferrricyanide and a separation voltage of 10 kV. In order to perform the FASS, samples were electrokinetically injected for 20 s at 20 kV into the capillary that was pre-field with a 4 s water plug. Analysis was performed at ambient temperature (22 ± 1 °C). The method’s validation revealed good linearity with respect to peak area ratios of MOR and MOR-6-G with the IS and the corresponding concentrations over the ranges of 1–2000 and 1.2 to 2000 ng/mL, respectively. Following one oral dose of controlled-release MOR sulphate tablet, the validated in-Cap-D-FASS-CZE method successfully enabled the determination of MOR and MOR-6-G in clinical serum samples.

Simultaneous Determination of Nicotine and Phenolic Compounds in Tobacco by Capillary Electrophoresis with Pipette Tip Electrodes

Aim: The aim of this work is to fabricate pipette tip electrodes for the capillary electrophoretic determination of nicotine and phenolic compounds in tobacco. Background: The content of nicotine affects not only the quality of tobacco products but also the health of smokers. Phenolic compounds are important flavor precursors in tobacco. The quantity of phenolic compounds is one of the most important evaluation indicators of tobacco quality. It is of high importance to determine nicotine and phenolic compounds in tobacco for quality control and the health of smokers. Objective: A method based on capillary electrophoresis and amperometric detection was developed for the simultaneous determination of nicotine, rutin, chlorogenic acid, quercetin, ferulic acid, gallic acid, and protocatechuic acid in tobacco leaves. Pipette electrodes were designed and fabricated for their amperometric detection. Method: Nicotine, rutin, chlorogenic acid, quercetin, ferulic acid, gallic acid, and protocatechuic acid were determined by capillary electrophoresis in combination with the detection electrodes that were fabricated by packing the composite of carbon nanotube and epoxy in pipette tips. Results: Detection potentials, the acidity and concentrations of background electrolyte, separation voltages, and injection times were optimized. At a high voltage of 12 kV, separation of the seven analytes could be achieved in less than 11 min in a piece of 40 cm long fused silica capillary with a background electrolyte of 50 mM borate buffer (9.2). Linearity was observed between the peak currents and the concentrations, with the limits of detection ranging from 0.1 to 0.2 μM for the seven analytes at the pipette electrodes. The method was applied in the simultaneous determination of nicotine and phenolic compounds with satisfactory assay results. Conclusions: The pipette tip electrodes were successfully coupled with capillary electrophoresis for tobacco analysis. The CE-AD method provides not only a simple approach for the quality control of tobacco and its preparations but also an alternative technique for the constituent and fingerprint investigation of other plants.

Inexpensive and simple tool for quality control of nutraceutical and tonic products with capillary electrophoresis and contactless conductivity detection: Some developments in Vietnam

In this study, we report the development of versatile and cost-effective analytical approaches for analysis and quality control of different nutraceutical and tonic products, using capillary electrophoresis (CE) coupled with contactless conductivity detection (C4D). Insights into CE-C4D instrumental designs that are adapted to the context in developing countries are provided. A highlight was made for quality control of vitamins, glucosamine and some minerals in different nutraceutical formulations collected from pharmacies in Vietnam. The lowest detection limit (LOD) achieved with the developed methods was 0.1 mg/L for vitamins, using the optimized background electrolyte (BGE) composed of 12 mM arginine (Arg)/acetic acid (AcOH) at pH 7.5 with 10 % acetonitrile (ACN). For magnesium and B6, the optimized CE-C4D conditions with the BGE composed of 10 mM Arg/AcOH (pH 5) with 20 % ACN allows its detection down to 0.05 mg/L. For glucosamine and calcium, the best performance with LOD of 0.05 mg/L was achieved with BGE composed of 10 mM Tris/AcOH (pH 5). Good agreement between results from CE-C4D and the confirmation methods was achieved for all investigated compounds, with their result deviations less than 15 %.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

Genetic diversity and population structure of Dermatophagoides farinae based on microsatellite markers

ABSTRACT Dermatophagoides farinae is one of the most important allergens that cause allergic diseases and trigger serious harm to the human body. D.farinae is widely distributed, and the development of genetic markers and genetic information regarding the populations of this species are needed to establish control strategies. The aim is to develop a microsatellite marker and assess the genetic diversity and population structure of different populations of D. farinae. The genomic DNA of D. farinae was extracted and sequenced by NGS analysis. Microsatellite markers were identified and screened with MISA software. Primers were designed with Primer5.0 and screened by capillary electrophoresis detection. Genotyping was implemented with GeneMarker 2.2. Genetic diversity and population structure were analysed by related softwares. Twelve microsatellite markers detected 66 alleles in 256 individuals of D. farinae from 12 geographic populations. The average allele number (Na), effective allele number (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) were 4.431, 2.610, 1.056 and 0.484, respectively. Genetic variation existed within populations and there was no correlation between geographic distance and genetic distance (Fst) (P = 0.160). Structure analysis showed D. farinae is largely divided into three groups. The genetic diversity of the geographical population of D. farinae is relatively high.

Validated Method for the Determination of Five Membered Heterocyclic Polar Compound Imidazole in Drug Substances Using Capillary Electrophoresis and UV Detection

A simple and sensitive method based on capillary electrophoresis with UV detection was developed, optimized, and validated for the determination of five membered heterocyclic polar compound, imidazole in pharmaceutical drug substances. Separation was achieved on bare fused silica capillary using a simple running buffer, potassium dihydrogen phosphate at pH 4.0. Capillary temperature set at 25°C. The applied voltage was 25 kV across the capillary and the samples were injected by hydrodynamically at a pressure of 50 mbar for 5 s. Analyte was monitored at 210 nm. The achieved limit of detection value was 0.005%w/w, limit of quantification value was 0.014%w/w, and the average accuracy value was 98.4% for imidazole. The aim of present study is to develop a specific and sensitive method for the determination of imidazole to overcome the void volume and sample matrix interferences.

A comparative study of commercial real-time reverse transcription PCR kits for forensic body fluid identification

ABSTRACT Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant (p > 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays.

Population Structure and Genetic Diversity Analysis in Sugarcane (Saccharum spp. hybrids) and Six Related Saccharum Species

Sugarcane (Saccharum spp. hybrids) is one of the most important commercial crops for sugar, ethanol, and other byproducts production; therefore, it is of great significance to carry out genetic research. Assessing the genetic population structure and diversity plays a vital role in managing genetic resources and gene mapping. In this study, we assessed the genetic diversity and population structure among 196 Saccharum accessions, including 34 S. officinarum, 69 S. spontaneum, 17 S. robustum, 25 S. barberi, 13 S. sinense, 2 S. edule, and 36 Saccharum spp. hybrids. A total of 624 polymorphic SSR alleles were amplified by PCR with 22 pairs of fluorescence-labeled highly polymorphic SSR primers and identified on a capillary electrophoresis (CE) detection system including 109 new alleles. Three approaches (model-based clustering, principal component analysis, and phylogenetic analysis) were conducted for population structure and genetic diversity analyses. The results showed that the 196 accessions could be grouped into either three (Q) or eight (q) sub-populations. Phylogenetic analysis indicated that most accessions from each species merged. The species S. barberi and S. sinense formed one group. The species S. robustum, S. barberi, S. spontaneum, S. edule, and sugarcane hybrids merged into the second group. The S. officinarum accessions formed the third group located between the other two groups. Two-way chi-square tests derived a total of 24 species-specific or species-associated SSR alleles, including four alleles each for S. officinarum, S. spontaneum, S. barberi, and S. sinense, five alleles for S. robustum. and three alleles for Saccharum spp. hybrids. These species-specific or species-associated SSR alleles will have a wide application value in sugarcane breeding and species identification. The overall results provide useful information for future genetic study of the Saccharum genus and efficient utilization of sugarcane germplasm resources in sugarcane breeding.

Influence of Organic Solvents and β-cyclodextrins on Capillary Zone Electrophoresis Separation of Five Biogenic Amines and Two B Vitamins

Abstract The effects of organic modifiers—alcohols (methanol, isopropanol), acetonitrile, and tetrahydrofuran—and β-cyclodextrins in capillary zone electrophoresis were investigated using a test mixture containing five biogenic amines important from the human health point of view—serotonin, dopamine, adrenaline, noradrenaline, and tyramine—and two B vitamins—thiamine and pyridoxine. The simultaneous addition of tetrahydrofuran and isopropanol was found to improve the resolution of determined analytes and enable effective separation of analytes with very similar electromigration characteristics migrating as one peak (dopamine and serotonin). The developed and optimised separation method based on capillary zone electrophoresis and ultraviolet detection was capable to achieve detection limits at the concentration level in the range of 0.15 to 1.25 μg/mL−1. The developed method was also characterised by other favourable validation parameters, such as linearity (r 2 &gt; 0.99), accuracy (82.9–117.8% for the intraday measurements, and 87.6–119.2% for the interday measurements), and precision (intraday relative standard deviation in the range of 0.4–15.5%, interday relative standard deviation in the range of 0.9–18.3%). The method was finally applied to investigate the stability of the analytes in model water matrix samples under various storage conditions.

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes.

ABSTRACTIt has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea.IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

A proof-of-principle study on implementing polymerase chain displacement reaction (PCDR) to improve forensic low-template DNA analysis

Polymerase chain reaction (PCR) plays an important role in forensic DNA analysis. However, the amplification of low-template DNA (LTDNA) samples usually encounters unsatisfactory results for the limited efficiency of PCR, which would interfere with the subsequent profile interpretation. Polymerase chain displacement reaction (PCDR) is a highly-efficient technique characterized by combining PCR and strand displacement reaction into a single PCDR cycle. This study explored the feasibility of PCDR for improving forensic LTDNA analysis. STR markers commonly used in forensic genetics were subjected to PCDR amplification and capillary electrophoresis detection. The results of singleplex reactions indicated that PCDR surpassed original PCR in efficiency for STR amplification. The average peak height of alleles in PCDR profiles was linearly correlated to the number of outer primers adopted for initiating the strand displacement process. Further, we assessed the multiplexing potential of PCDR by incorporating 17 STRs included in the expanded CODIS core loci and Amelogenin gene into a multiplex PCDR system. For pristine DNA templates ranged from 200 pg to 12.5 pg, the multiplex PCDR system consistently exhibited higher allele peak height as well as less allele dropout compared to the multiplex PCR references. Meanwhile, a significant reduction of stutter ratio was extensively observed in PCDR profiles. We also tested mock casework samples to verify the practical ability of multiplex PCDR for LTDNA detection. With DNA input varying from 48.1 pg to 6.6 pg, the multiplex PCDR system consistently obtained more allelic information than multiplex PCR methods. Our data collectively suggested that it is feasible to apply PCDR in forensic LTDNA analysis.