Capillary Electrophoresis Method Research Articles

Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion.

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

Development of a novel capillary electrophoresis method for quantitative measurements of intracellular recombinant protein titer

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb.

Advance Herbal Technology

Lately peoples are getting attracted towards herbal medicines due to many benefits. Herbal formulations have achieved extensive acceptability as medicinal agents for several diseases. Although, most of these applications are unnatural, it is however a known truth that over 80% of the world population depends on herbal medicines and product for healthful living. This is a development in the use of herbal product has again given height to various forms of misuse and impurity of the products leading to consumers’ and manufacturers’ dissatisfaction and in some instances fatal effects. The development of original analytical methods which can reliably profile the phytochemical arrangement, including quantitative analyses of marker/bioactive compounds and other major components, is a major challenged to scientists. Standardization is an important step for the organization of a consistent biological activity, a consistent chemical profile, or just a quality confirmation program for exposition and manufacturing of herbal drugs. In current review essay various convectional techniques as well as newer advances are described. Recent advancements includes DNA fingerprinting, metabolomics method, differintial vibration polarography, chemometric, X-ray diffraction…etc. are practical. Capillary electrophoresis and chromatographic methods contributions towards standardization of herbal drugs is also documented.

Исследование аналитических возможностей метода капиллярного электрофореза для определения неорганических анионов в спиртных дистиллированных напитках

Содержание отдельных анионов в зависимости от концентрации может оказывать как положительное, так и отрицательное влияние на органолептические характеристики алкогольных напитков. В работе исследована возможность использования метода капиллярного электрофореза в целях изучения анионного состава спиртных дистиллированных напитков. Для проведения исследований выбрана система капиллярного электрофореза PrinCE, обладающая необходимыми техническими характеристиками. В ходе исследований приготовлены рабочие растворы хлоридов, нитратов и сульфатов массовой концентрацией 0,1 мг/дм и 20,0 дм, соответствующие началу и концу предполагаемого диапазона измерений. Экспериментально определены лучшие рабочие параметры системы капиллярного электрофореза, необходимые для достижения оптимального разделения и чувствительности. Проведенные эксперименты показали, что полное разрешение сигналов хлорид-, нитрат- и сульфат-ионов возможно при следующих условиях: длина и диаметр кварцевого капилляра 40 см и 50 мкм соответственно, напряжение +30 кВ, инжектирование гидродинамическим способом, давлением ввода 35 мБар, время ввода 20 сек, температура капилляра +25 °С, детектирование кондуктометрическое. Определены последовательность и время выхода пиков целевых анионов, построены калибровочные графики, подтверждена линейность, разработана схема промывки капилляра, обеспечивающая стабильность времени миграции. Установлено, что погрешность определения хлорид-, нитрат- и сульфат-ионов предлагаемым способом не превышает 20 %. Для подтверждения применимости описанной электрофоретической процедуры в целях определения ионов неорганической природы в спиртных дистиллированных напитках проанализировано более 25 типичных образцов. Диапазон массовых концентраций определяемых ионов в исследованных образцах колебался в широких пределах: для хлорид-ионов от 0,57 до 18,25 мг/дм, для нитрат-ионов 0,08-3,33 мг/дм, для сульфат-ионов 0,29-8,57 мг/дм. Выявлена некоторая закономерность: в составе исследованных образцов виски превалировали хлорид- и сульфат-ионы, в составе образцов рома, текилы и самогона преобладали сульфат-ионы. Экспериментально подтверждено, что разработанная аналитическая процедура применима для экспрессного селективного определения хлорид-, нитрат-, сульфат-ионов за 4 мин в алкогольных напитках методом капиллярного электрофореза с кондуктометрическим детектированием. Предлагаемый подход может быть рекомендован для разработки метрологически аттестованной методики определения ионов неорганической природы в спиртных напитках. The content of individual anions, depending on the concentration, can have both positive and negative effects on the organoleptic characteristics of alcoholic beverages. The possibility of using the method of capillary electrophoresis in order to study the anionic composition of distilled alcoholic beverages was studied in this work. The PrinCE capillary electrophoresis system, which has the necessary technical characteristics, was chosen for the research. In the course of the research, working solutions of chlorides, nitrates and sulfates were prepared, with a mass concentration of 0.1 mg/dm and 20.0 dm, corresponding to the beginning and end of the proposed measurement range. The best operating parameters of the capillary electrophoresis system necessary to achieve optimal separation and sensitivity have been experimentally determined. The experiments performed have shown that the complete resolution of the signals of chloride, nitrate and sulfate ions is possible under the following conditions: the length and diameter of the quartz capillary are 40 cm and 50 μm, respectively, voltage +30 kV, hydrodynamic injection, injection pressure 35 mbar, time input 20s, capillary temperature +25 °C, conductometric detection. The sequence and exit times of target anion peaks were determined, calibration graphs were constructed, linearity was confirmed, and a capillary flushing scheme was developed to ensure the stability of migration times. It has been established that the error in determining chloride-nitrate-sulfate ions by the proposed method does not exceed 20 %. To confirm the applicability of the described electrophoretic procedure for the determination of inorganic ions in distilled alcoholic beverages, more than 25 typical samples were analyzed. The range of mass concentrations of the analyzed ions in the studied samples varied over a wide range: for chloride ions from 0.57 to 18.25 mg/dm, for nitrate ions 0.08- 3.33 mg/dm, for sulfat ions 0.29-8.57 mg/dm. A certain pattern was revealed: chloride and sulfate ions prevailed in the composition of the studied whiskey samples, sulfate ions prevailed in the composition of rum, tequila and moonshine samples. It has been experimentally confirmed that the developed analytical procedure is applicable for the rapid selective determination of chloride, nitrate, and sulfate ions in alcoholic beverages by capillary electrophoresis with conductometric detection. The proposed approach can be recommended for the development of a metrologically certified method for the determination of inorganic ions in alcoholic beverages.

A Capillary Zone Electrophoresis Method for Selenite Detection in Culture Media for Total Selenium Determination in Yeast Cells

Selenium enriched yeast is the most prevalent source of organic selenium for humans and animals. In this study, an effective capillary zone electrophoresis method was established to quantify total selenium in yeast biomass by measuring selenite level in post incubated culture media. The detection of selenite could be achieved within 7.9 min under the condition of a mixture buffer of 85 mM Na2HPO4 and 0.5 mM CTAB (pH 9.37), –5 kV separation voltage with 1.0 psi × 30 s injection at 200 nm. The range of calibration curve linearity of the standard was from 5 to 300 mg/L (r2 = 0.99998), and the recovery was 102.4%. Owing to the ease of sample collection and simple detection, this method is convenient for the evaluation of total selenium accumulated in yeast cell biomass and helps to guide the production of selenium enriched yeast.

Advanced Herbal Technology

Due to their numerous benefits, herbal medications are increasingly attracting people's attention. As treatment options for many disorders, herbal formulations have gained widespread acceptance. Over 80% of the world's population relies on herbal products and medications for a healthy lifestyle, despite the fact that the majority of these uses are unconventional. As the usage of herbal goods has increased, so have the misuse and adulteration of the products, which has disappointed customers and producers and, in some cases, had disastrous results. The creation of reliable analytical techniques that can profile the phytochemical composition with accuracy, including quantitative studies of marker/bioactive chemicals and other important elements, is a significant challenge for scientists. Standardization is a critical essential step in the development ofa quality assurance process, a consistent chemical profile, or simply a constant biological activity for the manufacture and manufacturing of herbal medications. The numerous convectional approaches and more recent advancements are covered in the current review article. Recent developments have been noted in the areas of DNA fingerprinting, metabolomics, differential pulse polarography, chemometrics, X-ray diffraction, etc. Contributions of chromatographic and capillary electrophoresis methods to the standardisation of herbal medicines are also described.

Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection.

The need for analytical methods that are fast, affordable, and ecologically friendly is expanding. Because of its low solvent consumption, minimal waste production, and speedy analysis, capillary electrophoresis is considered a "green" choice among analytical separation methods. With these "green" features, we have utilized the capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4D) to simultaneously determine glucosamine and Ca2+ in dietary supplements. The CE analysis was performed in fused silica capillaries (50 μm inner diameter, 40 cm total length, 30 cm effective length), and the analytical time was around 5 min. After optimization, the CE conditions for selective determination of glucosamine and Ca2+ were obtained, including a 10 mM tris (hydroxymethyl) aminomethane/acetic acid (Tris/Ace) buffer of pH 5.0 as the background electrolyte; separation voltage of 20 kV; and hydrodynamic injection (siphoning) at 25 cm height for 30 s. The method illustrated good linearity over the concentration range of 5.00 to 200 mg/L of for glucosamine (R 2 = 0.9994) and 1.00 to 100 mg/L for Ca2+ (R 2 = 0.9994). Under the optimum conditions, the detection limit of glucosamine was 1.00 mg/L, while that of Ca2+ was 0.05 mg/L. The validated method successfully analyzed glucosamine and Ca2+ in seven dietary supplement samples. The measured concentrations were generally in line with the values of label claims and with cross-checking data from reference methods (HPLC and ICP-OES).

Development of Capillary Zone Electrophoresis Method for the Simultaneous Separation and Quantification of Metformin and Pioglitazone in Dosage Forms; and Comparison with HPLC Method.

A capillary zone electrophoretic (CZE) method was developed, validated, and applied for the assay of metformin (MET) and pioglitazone (PIO) in pharmaceutical formulations. The optimum running buffer composition was found to be 75 mmol/L phosphate buffer containing 30% acetonitrile (ACN) at pH 4.0. The optimum instrumental conditions were found to be injection time, 10 s; applied voltage, 25 kV; hydrodynamic injection pressure, 0.5 psi for 10 s, capillary temperature, 25 °C; and the detection wavelength, 210 nm. The quantifications were calculated based on the ratio of the peak areas of analytes to atenolol as an internal standard. The CZE method was validated in terms of accuracy (98.21-104.81%), intra- and inter-day precision of migration time and peak area (relative standard deviation ≤ 5%), linearity (correlation coefficients ≥ 0.9985), limit of detection (≤0.277 μg/mL), and limit of quantitation (≤0.315 μg/mL). The proposed method was applied for the analysis of PIO and MET both individually and in a combined dosage tablet formulation. All electrophoretic parameters were calculated and evaluated. A previously reported high-performance liquid chromatographic (HPLC) method was also applied to the same samples. A comprehensive comparison was then carried out for the analytical features of both methods CZE and HPLC. Comparable results were obtained with the advantage of reagent consumption and separation efficiency of CZE over HPLC and shorter analysis time by HPLC compared with CZE.

Plasma levels of creatine, 2-aminobutyric acid, acetyl-carnitine and amino acids during fasting measured by counter-current electrophoresis in PAMAPTAC capillary

Determination of the minor nitrogen metabolites, creatine, 2-aminobutyric acid (2-AB), and acetyl-carnitine (Ac-carn), in human plasma is performed by capillary electrophoresis (CE) in a 6 % PAMAPTAC coated fused silica capillary. 6 % PAMAPTAC generates a stable electroosmotic flow (EOF) of 6.31 × 10−9 m2V−1s−1; and the nitrogen metabolites are separated in a counter-current mode, which increases electrophoretic resolution. Baseline separation of creatine, 2-AB, Ac-carn, and most proteinogenic amino acids is achieved in 8.5 M acetic acid (AcOH) at pH 1.37 as background electrolyte (BGE) at an effective capillary length of 14.4 cm with migration times ranging from 4.95 min for creatine to 5.43 min for Ac-carn. 20 μL of plasma is deproteinized with the addition of 60 μL of acetonitrile (ACN) and a 21.8 mm sample zone is injected into the capillary, which corresponds to 6.9 % of total and 15 % of effective capillary length. The zone is focused using a large volume sample stacking technique with LODs of 0.2–0.4 μM and LOQs of 0.8–1.3 μM relative to the untreated plasma sample. Inter-day reproducibility of migration time is 0.7–1.1 % and for peak area 2.5–3.9 %. The CE method is used to monitor plasma levels of nitrogen metabolites during 60 h of fasting in 10 healthy lean volunteers. T-test showed statistically significant differences in plasma levels of creatine, 2-AB, Ac-carn and most proteinogenic amino acids, which return to their original concentrations after subsequent 48 h recovery.

Application of microfluidic chip electrophoresis for high-throughput nucleic acid fluorescence fragment analysis assays.

Nucleic acid fragment analysis via separation and detection are routine operations in molecular biology. However, analysis of small single-stranded nucleic acid fragments (<100nt) is challenging and mainly limited to labor-intensive polyacrylamide gel electrophoresis or high-cost capillary electrophoresis methods. Here we report an alternative method, a microfluidic chip electrophoresis system that provides a size resolution of 5nt and a detection time of one minute per sample of fluorescence-labeled DNA/RNA fragments. The feasibility of this system was evaluated by quantifying CRISPR-Cas9 cleavage efficiency and the detection resolution was evaluated by analyzing ssDNA/RNA adenylation and phosphorylation. We employed this system to study the RNA capping efficiency and double-stranded DNA unwinding efficiency in isothermal amplification as two examples for assay design and evaluation. The microfluidic chip electrophoresis system provides a rapid, sensitive, and high-throughput fluorescence fragment analysis (FFA), and can be applied for enzyme characterization, reaction optimization, and product quality control in various molecular biology processes.

Direct count of fluorescent microspheres in a microfluidic chip based on the capillary electrophoresis method.

Fluorescent microspheres (FMs) are tiny particles with special functions that are widely employed in biological research. Counting of microscale FMs is a great challenge by capillary electrophoresis. Herein we developed a method to count 2 μm FMs based on a microfluidic chip with a gradual change in inner size. Such a microfluidic chip can inhibit sample blocking at the inlet of the capillary. The results showed that FMs migrated in the wide part of the microchannel side by side, and then passed through the narrow part one by one. There was a linear relationship between the number of peaks in the electropherogram and concentration of FMs if they were running in the microchannel for more than 20 min. A high separation voltage may lead to aggregation of FMs in the microchannels, and about 2 × 104 FMs can be counted within 30 min by this microfluidic chip.

Bioactive peptides of amaranth: isolation and determination of antioxidant and hypocholesterolemic properties

Hydrolysis of the amaranth protein isolate (Amaranthus hypochondriacus L., Voronezh variety) protein isolate by trypsin, papain and alkalase was carried out in order to obtain biologically active peptides with antioxidant and hypocholesterolemic activities. A high degree of protein hydrolysis was shown by trypsin during the first two hours, papain between three and four hours, and alkalase after four hours of hydrolysis. The rate of cleaved protein increased from the first two hours of hydrolysis from 40%, 30% and 15% to 78%, 70% and 60% when trypsin, papain, alkalase were applied, respectively. Maximum hydrolysis of the amaranth protein was observed after 6 h of proteolysis by all enzyme preparations. The molecular weights of the hydrolysis products of the amaranth protein after 2, 4 and 6 h of hydrolysis were determined. All hydrolysates showed a high content of polypeptides with molecular masses of 33 kDa, 18-23 kDa, 52-54 kDa, 40-54 kDa, 6.5-15 kDa. Short-chain peptides with molecular masses of 624 to 2817 Da were detected in the hydrolysates after 6 h of proteolysis. It was determined by capillary electrophoresis method that all protein hydrolysates after 6 h of hydrolysis contain amino acids (tyrosine, phenylalanine, alanine, leucine, valine, proline, alanine) responsible for antioxidant activity of peptides in the studied hydrolysates. The antioxidant activity of peptides in all hydrolysates of amaranth protein isolate was determined by ABTS cation radical reduction method for 6 min. Hypocholesterolemic activity of peptides was shown in protein hydrolysates by trypsin and papain.

Fast and Environmentally Friendly Method for Simultaneous Determination of Hydrochlorothiazide, Losartan and Potassium by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

Losartan potassium and hydrochlorothiazide are often combined in pharmaceutical formulations for the treatment of hypertension. Therefore, the determination of these compounds in a single run is highly desirable for rapid quality control applications. The present study describes an ultra-fast (ca. 85 injections h−1) and environmentally friendly method based on capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection for simultaneous quantification of potassium, losartan and hydrochlorothiazide. Cation (potassium) and anions (losartan and hydrochlorothiazide) were analyzed in a single run using a background electrolyte composed by 10.0 mmol L-1 boric acid (pH = 9.0, adjusted with sodium hydroxide). The limits of detection were 4.0, 3.0 and 10.0 μmol L-1 for potassium, losartan and hydrochlorothiazide, respectively. The proposed method is simple, fast, with minimal waste generation, and accurate (recovery values between 98 and 102%). The results obtained with the CE method were statistically similar (95% confidence level) to those obtained by high-performance liquid chromatography (losartan and hydrochlorothiazide) and flame photometry (potassium).

Evaluation strategy of drug permeability in new drug discovery and development stage

Drug permeability is an important factor determining the effectiveness and safety of drug treatment. In the early stage of new drug research and development, testing drug permeability can improve research and development efficiency and reduce research and development costs. Because of its advantages such as rapidity and simplicity, drug permeability has gradually attracted attention. This paper introduces the methods of two-phase distribution, chromatography, capillary electrophoresis, optical sensor and parallel artificial membrane permeation test at home and abroad in recent years.

Advance herbal technology

Lately peoples are getting attracted towards herbal medicines due to many benefits. Herbal formulations have achieved extensive acceptability as medicinal agents for several diseases. Although, most of these applications are unnatural, it is however a known truth that over 80% of the world population depends on herbal medicines and product for healthful living. This is a development in the use of herbal product has again given height to various forms of misuse and impurity of the products leading to consumers’ and manufacturers’ dissatisfaction and in some instances fatal effects. The development of original analytical methods which can reliably profile the phytochemical arrangement, including quantitative analyses of marker/bioactive compounds and other major components, is a major challenged to scientists. Standardization is an important step for the organization of a consistent biological activity, a consistent chemical profile, or just a quality confirmation program for exposition and manufacturing of herbal drugs. In current review essay various convectional techniques as well as newer advances are described. Recent advancements includes DNA fingerprinting, metabolomics method, differintial vibration polarography, chemometric, X-ray diffraction…etc. are practical. Capillary electrophoresis and chromatographic methods contributions towards standardization of herbal drugs is also documented.

THE EVALUATION OF TERTIARY CARE CENTER HEMOGLOBIN VARIANT DATA FOR THREE YEARS PERIOD

Objective: Hemoglobin disorders are one of the most common hereditary diseases in the world. In this study, we aimed to evaluate the hemoglobin variants detected by hemoglobin electrophoresis for a three years period in a tertiary care center. Materials and Methods: Hemoglobin variant analysis results of 4804 different variants for a three years period were evaluated retrospectively. Hemoglobin variant analysis was performed by capillary electrophoresis method on Minicap Flex Piercing analyzer (Sebia, Lisses, France). Results: One thousand and six (20.94%) of hemoglobin variants were detected in the study. The number of the patients with thalassemia trait was 1028 (21.39%) and the number of the patients with Beta thalassemia major was 44 (0.91%). In the study, the most common hemoglobin variant was found to be HbF (45.72%). The other hemoglobin variants in decreasing order were HbD, HbS, HbE, HbC and HbH. Conclusion: The place where the study was conducted is not the region where hemoglobinopathy is most commonly known in our country. However, the outcomes of the study indicated different results than expected. It should be noted that the frequency of hemoglobinopathy and the regions where it is seen frequently may change due to the migration wave that occurs due to reasons such as sociocultural, economic and war conditions.

Evaluation of interference from 16 hemoglobin variants on hemoglobin A1c measurement by five methods

Hb variants prevalent in China are different from those in other countries. We aimed to assess the interference from Hb variants found in China on HbA1c measurement. All Hb variants were confirmed using Sanger sequencing. HbA1c was measured using a capillary electrophoresis method (Capillarys 3 OCTA), two cation-exchange high-performance liquid chromatography methods (ADAMS HA-8180V and HLC-723 G8 standard mode), an immunoassay (Cobas c501), and a boronate affinity chromatography method (Premier Hb9210). Premier Hb9210 was used as a comparative method. A total of 16 species of Hb variants were identified in 102 variant carriers. The most common variant was Hb E, followed by Hb Q-Thailand, Hb New York and Hb J-Bangkok. Clinically significant interference was observed for the Capillarys 3 OCTA (two Hb variants), ADAMS HA-8180V (seven Hb variants), HLC-723 G8 (14 Hb variants), and Cobas c501 (two Hb variants). The proportion of unacceptable HbA1c results was 13.7% for Capillarys 3 OCTA, 52.9% for HA-8180V, 83.3% for HLC-723 G8, and 3.9% for Cobas c501. Hb variants in China severely affect the accuracy of some commonly used HbA1c methods.

Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

Enzyme-Linked Aptamer Kits for Rapid, Visual, and Sensitive Determination of Lactoferrin in Dairy Products.

Lactoferrin (Lf), as a popular nutritional fortification in dairy products, has the ability regulate the body's immune system and function as a broad-spectrum antibacterial, which is of great significance to the growth and development of infants and children. Herein, an indirect competitive enzyme-linked aptamer assay (ELAA) kit was established for rapid, sensitive, and visual determination of Lf in dairy products. In the construction, the Lf aptamer was conjugated with horseradish peroxidase (HRP) as the recognition probe and aptamer complementary strand (cDNA) were anchored onto the microplate as the capture probe. The recognition probes were first mixed with a sample solution and specifically bound with the contained Lf, then added into the microplate in which the free recognition probes in the mixture were captured by the capture probe. After washing, the remaining complex of cDNA/Aptamer/HRP in the microplate was conducted with a chromogenic reaction through HRP, efficiently catalyzing the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB), therefore the color shade would directly reflect Lf concentration. Under the optimization conditions, a good linear relationship (R2, 0.9901) was obtained in the wide range of 25-500 nM with the detection limit of 14.01 nM and a good specificity, as well as the reliable recoveries. Furthermore, the ELAA kits achieved the Lf determination with an accuracy of 79.71~116.99% in eleven samples, which consisted of three kinds of dairy products: including goat milk powder, cow milk powder, and nutrition drop. Moreover, the results were also validated by the high-performance capillary electrophoresis (HPCE) method. The ELAA kit provides a simple and convenient determination for Lf in dairy products, and it is highly expected to be commercialized.

Evaluation of loci to predict ear morphology using two SNaPshot assays.

Human ear morphology prediction with SNP-based genotypes is growing in forensic DNA phenotyping and is scarcely explored in Pakistan as a part of EVCs (externally visible characteristics). The ear morphology prediction assays with 21 SNPs were assessed for their potential utility in forensic identification of population.The SNaPshot™ multiplex chemistries, capillary electrophoresis methods and GeneMapper™ software were used for obtaining genotypic data. A total of 33 ear phenotypes were categorized with digital photographs of 300 volunteers. SHEsis software was applied to make LD plot. Ordinal and multinomial logistic regression was implemented for association testing. Multinomial logistic regression was executed to construct the prediction model in 90% training and 10% testing subjects.Several influential SNPs for ear phenotypic variation were found in association testing. The model based on genetic markers predicted ear phenotypes with moderate to good predictive accuracies demonstrated with the area under curve (AUC), sensitivity and specificity of predicted phenotypes.As an additional EVC, the estimated ear phenotypic profiles have the possibility of determining the human ear morphology differences in unknown biological samples found in crimes that do not result in a criminal database hit. Furthermore, this can help in facial reconstruction and act as an investigational lead.