Search Results (64)

This study aimed to develop a reliable method for profiling reducing sugars in honey using capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). Reducing sugars were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in the presence of 2-picoline borane, a safer alternative to sodium cyanoborohydride. Key parameters influencing the derivatization efficiency—temperature, pH, incubation time, and reagent concentrations—were systematically optimized. The highest labeling efficiency for glucose, mannose, and maltose was achieved at 50 °C in 0.5 M citric acid with 0.1 M APTS, while fructose showed low reactivity due to its ketose structure. To reduce the background signal from excess reagents, three cleanup strategies were evaluated. Liquid–liquid extraction with ethyl acetate effectively removed unreacted APTS without significant analyte loss, whereas solid-phase extraction and microextraction caused substantial losses of hydrophilic sugars. The method showed good linearity (0.5–10 mM, R² > 0.994), precision (RSD 0.81–13.73%), and accuracy (recoveries 93.47–119.75%). Stability studies indicated that sugar standards should be stored at –20 °C. The method was successfully applied to the analysis of four nectar honeys—rapeseed, acacia, phacelia, and dandelion—revealing differences in glucose and fructose content related to botanical origin. The results confirm the suitability of CZE-LIF for sensitive and selective carbohydrate analyses in complex food matrices. Full article

The stability of collagen, the most abundant protein in humans and many animals, is related to the hydroxylation of L-proline, a post-translational modification occurring at carbon 3 and 4 on its pyrrolidine ring. Collagens of different origins have shown different proline hydroxylation levels, making hydroxyprolines useful biomarkers in structure characterizations. The presence of two chiral carbon atoms, 3-hydroxyproline and 4-hydroxyproline, results in eight stereoisomers (four pairs of enantiomers) whose quantitation in collagen hydrolysates requires enantioselective analytical methods. Capillary electrophoresis was applied for the separation and quantitation of the eight stereoisomers of 3- and 4-hydroxyproline and D,L-proline in collagen hydrolysates. The developed method is based on the derivatization with the chiral reagent (R)-(-)-4-(3-Isothiocyanatopyrrolidin-yl)-7-nitro-2,1,3-benzoxadiazole, enabling the use of a light-emitting diode-induced fluorescence detector for high sensitivity. The separation of the considered compounds was accomplished in less than 10 min, using a 500 mM acetate buffer pH 3.5 supplemented with 5 mM of heptakis(2,6-di-O-methyl)-β-cyclodextrin as the chiral selector. The method was fully validated and showed the adequate sensitivity for the application to samples of collagen hydrolysates. The analysis of samples extracted from chicken Pectoralis major muscles affected by growth-related myopathies showed different stereoisomer patterns compared to those from the unaffected control samples. Full article

Background: FLT3 mutation testing is a key ancillary molecular assay for diagnosing and managing patients with acute myeloid leukemia (AML), including assessing the utility of FLT3 inhibitors during induction chemotherapy. FLT3 PCR utilizing fluorescently labeled primers and capillary electrophoresis readout is the most used technique for the rapid detection of FLT3 internal tandem duplications (ITDs) (including very small ITDs) and tyrosine kinase domain (TKD) mutations. However, capillary electrophoresis (CE) is a relatively lengthy and technically demanding result readout mode that could potentially be replaced by faster alternatives. Methods: Here, we describe the validation of a modified FLT3 PCR assay that uses the Agilent 4200 TapeStation platform for result readouts. This platform generates quantifiable electropherograms and gel images in under two minutes and at a low cost. We validated its ability to detect FLT3-ITD and -TKD mutations using 22 and 18 previously tested patient samples, respectively. Results: The TapeStation 4200 instrument is 100% sensitive, specific, and highly reproducible for post-PCR fragment analysis in detecting FLT3-ITD (greater than 15 bp in size) and TKD mutations in AML patients. Its results are nearly 100% concordant with those obtained from our previously validated NGS and PAGE methods. However, the limitation of this readout mode is its inability to reliably detect FLT3-ITDs smaller than 15 bp in size. Conclusions: Given the widespread use of TapeStation instruments in molecular diagnostics laboratories as part of next-generation sequencing (NGS) workflows, this modified assay is well-suited as a companion test for rapid NGS platforms to detect larger FLT3-ITDs, which are NGS often miscalledor under-called by the NGS bioinformatics algorithms. However, it is not suitable for use as a standalone assay, as it is unable to reliably detect very short FLT3-ITDs. Full article

Capillary electrophoresis based on laser-induced fluorescence (CE-LIF) plays an important role in the analysis of nucleic acids. However, the commercial CE-LIF is not only quite expensive but also inflexible, thus hindering its widespread use in the lab. Herein, we proposed a compact, low-cost, and flexible CE-LIF system. We also investigated its stability by separating the DNA ladders. Experiments demonstrated that the relative standard error of the relative fluorescence intensity and migration time was lower than 6.2% and 1.1%, respectively. The aperture size of the light source illuminating the capillary can affect the separation performance. Smaller apertures offer higher resolution length for the adjacent DNA fragments but may reduce the number of theoretical plates. Various fluorescent dyes (e.g., SYBR Green I, Gel Green, EvaGreen) can be employed in the self-built system. The limit of detection of dsDNA was as low as 0.05 ng/μL. The working range for DNA was 0.05 ng/μL~10 ng/μL. Finally, we have successfully separated the PCR products of the target gene of Porphyromonas gingivalis and Candida albicans in the home-built CE system. Such a robust CE-LIF system is easy to assemble in the lab. The total cost of the assembled CE system did not exceed 1100 USD. We believe this work can advance the application of CE and hope it will facilitate the easy assembly of flexible CE instruments in labs. Full article

Introduction: Obesity is a rapidly growing common health problem worldwide that can lead to the development of gestational diabetes mellitus (GDM). However, GDM not only affects women with obesity but can also develop at any time, even after the OGTT test; therefore, an increasing number of complications related to GDM can be seen in both mothers and their children. It is necessary to discover biomarkers capable of indicating the development of GDM or complications during/after pregnancy. Since the N-glycosylation motif of human IgG has been described to change under many physiological and pathological conditions, it is a promising target for biomarker research. In our study, the effects of obesity and GDM were investigated on human serum IgG N-linked glycosylation patterns during human pregnancy. Materials and Methods: The study participants were categorized into four groups according to their body mass index (BMI) and GDM status: normal weight as control, obese (BMI > 30 kg/m²), normal weight with GDM, and obese with GDM. The released N-glycan components of IgG were separated with capillary electrophoresis and detected using a laser-induced fluorescence detector. Results: The result revealed several differences between the N-glycosylation patterns of the four study groups. Of this, 17 of the 20 identified structures differed significantly between the groups. The ratios of sialylated to non-sialylated structures were not changed significantly, but the core fucosylation level showed a significant decrease in the GDM and obese GDM groups compared to the control subjects. The lowest degree of core fucosylation was observed in the GDM group. Conclusions: The findings indicate that obesity in isolation does not have a significant impact on the IgG N-glycosylation pattern in pregnancy. Conversely, alterations in the N-glycan profile of antibodies may serve as biomarkers for the diagnosis of GDM in mothers with a normal BMI, although more evidence is needed. By incorporating glycan-based biomarkers into clinical practice, healthcare providers can improve early detection, personalize management strategies, and potentially mitigate adverse pregnancy outcomes associated with obesity and GDM. Full article

Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product. Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal. Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product. Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production. Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process. Full article

The early diagnosis of tumorigenesis is crucial for clinical treatment, but the resolution and sensitivity of conventional short-wavelength biomarkers are not ideal because of the complicated interference in living tissue. Herein, a nicotinamide adenine dinucleotide (NAD⁺)-responsive probe with deep-red emissive ratiometric fluorescence was synthetized as a promising target for energy metabolism patterns during tumorigenesis. Interestingly, the solvents H₃PO₄ and 2,2′-dithiodibenzoic acid enhanced the red emission (640 and 680 nm) of o-phenylenediamine-based carbon dots (CDs), leading to the formation of a nanoscale graphite-like skeleton covered with -P=O, -CONH-, -COOH and -NH₂ on their surfaces. Meanwhile, this method exhibited high sensitivity to the discriminating target NAD⁺, with a detection limit of 63 μM due to the inner filter effect and fluorescence resonance energy transfer process between NAD⁺ and CDs, which is superior to the reported capillary electrophoresis and liquid chromatographic detection methods (the reported detection limit was about 0.2 mM) in complex biological samples and even cancer cells. Encouragingly, NAD⁺ significantly promoted nucleus-targeting fluorescence and cell migration compared to GSH and pH stimulation, which were gradually eliminated in human hepatocellular carcinoma (HepG2) cells after 2-deoxy-d-Glucose inhibited the glycolytic phenotype. The proposed method holds great potential for the temporal and spatial resolution of NAD⁺-dependent tumor diagnosis in complex living systems. Full article

Globally, intron 22 inversions (Inv22s) of the factor VIII gene (F8) are the most frequent pathogenic variants and account for 45–50% of severe hemophilia A (SHA) cases, while intron 1 inversion (Inv1) explains 1–5% of SHA cases. The detection of both inversions by an inverse shifting-polymerase chain reaction (IS-PCR) is the first choice worldwide for the diagnosis of patients and carriers of SHA. To improve its sensitivity and reproducibility in the visualization of PCR products, we approached the IS-PCR with fluorescent capillary electrophoresis instead of agarose electrophoresis. Based on the original protocol, we modified two primers by 5’-end labeling with FAM™ fluorescent dye for the detection of the PCR products by capillary electrophoresis. Additionally, the “fast enzymes” BclI and T4-Ligase were incorporated for work saving in the genomic digestion and ligation reactions, respectively. Once we accomplished the standardization and verified the reproducibility of the modified IS-PCR method, we applied it for the diagnosis of a cohort of SHA patients and carriers. The modified IS-PCR by fluorescent capillary electrophoresis for PCR product detection is more sensitive than agarose electrophoresis. The method was also improved by using the new rapid enzymes to save time in the whole process. Full article

The significance and necessity of separating enantiomers in food, pharmaceuticals, pesticides, and other samples remains constant and unrelenting. The successful chiral separation usually includes the application of a chiral auxiliary compound, known also as a chiral selector (CS), that forms complexes with enantiomers of different physicochemical properties, enabling efficient separation. While both native and substituted cyclodextrins (CDs) are commonly used as CSs, β-CD is undoubtedly the most popular one among them. This review includes recent advancements in the application of β-CD as a CS. While the theoretical background behind the enantioseparation is also part of this work, the main emphasis is put on the factors that affect the efficacy of this process such as temperature, pH, solvent, and the choice of other additives. Also, the different analytical methods: Nuclear Magnetic Resonance (NMR) spectroscopy, Capillary Electrophoresis (CE), fluorescence spectroscopy (FS), High-Performance Liquid Chromatography (HPLC), Isothermal Titration Calorimetry (ITC), and UV–vis spectroscopy, used for enantioseparation with the aid of β-CD as CS, are thoroughly compared. Also, since some of the chiral compounds have been studied in the context of their enantioseparation more than once, those works are compared and critically analyzed. In conclusion, while β-CD can be in most cases used as CS, the choice of the experimental conditions and method of analysis is crucial to achieve the success. Full article

Gangliosides are sialic acid-containing glycosphingolipids that play an essential role in many biological and pathophysiological processes. They are present in high amounts in the central nervous system and their abnormal metabolism or expression has been observed in many diseases. We have developed and validated a sensitive capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the separation and quantification of oligosaccharides digested from nine gangliosides of high biological relevance. APTS was used for the labeling of the glycans. Reverse polarity CE was performed for the separation of the labeled glycans bearing negative charges. The optimized background electrolyte is a 15 mM lithium acetate buffer with pH of 5 containing 5% w/v linear polyacrylamide, which allows for the separation of all nine gangliosides. Validation parameters including linearity, precision, and accuracy were evaluated. LOQ and LOD were in the nM range, comparable to those of LC-MS techniques. The method was used to identify and quantify the ganglioside pattern of glioblastoma and neuroblastoma cell lines. The presented method is a valuable tool for further investigations aiming at understanding the role of gangliosides in various neurological diseases or CNS tumors. Full article

Phaseolus coccineus L. is a highly valuable crop for human consumption with a high protein content and other associated health benefits. Herein, 14 P. coccineus L. landraces were selected for genetic characterization: two Protected Geographical Indication (PGI) landraces from the Prespon area, namely “Gigantes” (“G”) and “Elephantes” (“E”), and 12 additional landraces from the Greek Gene Bank collection of beans (PC1–PC12). The genetic diversity among these landraces was assessed using capillary electrophoresis utilizing fluorescence-labeled Simple Sequence Repeat (SSR) and Expressed Sequence Tag (EST); Simple Sequence Repeat (SSR) is a molecular marker technology. The “G” and “E” Prespon landraces were clearly distinguished among them, as well as from the PC1 to PC12 landraces, indicating the unique genetic identity of the Prespon beans. Overall, the genetic characterization of the abundant Greek bean germplasm using molecular markers can aid in the genetic identification of “G” and “E” Prespon beans, thus preventing any form of fraudulent practices as well as supporting traceability management strategies for the identification of authenticity, and protection of the origin of local certified products. Full article

This study examines the intricate relationship between protein glycosylation dynamics and therapeutic responses in Luminal A and Luminal B breast cancer subtypes, focusing on anastrozole and tamoxifen impacts. The present methods inadequately monitor and forecast patient reactions to these treatments, leaving individuals vulnerable to the potential adverse effects of these medications. This research investigated glycan structural changes by following patients for up to 9 months. The protocol involved a series of automated steps including IgG isolation, protein denaturation, glycan labelling, purification, and final analysis using capillary gel electrophoresis with laser-induced fluorescence. The results suggested the significant role of glycan modifications in breast cancer progression, revealing distinctive trends in how anastrozole and tamoxifen elicit varied responses. The findings indicate anastrozole’s association with reduced sialylation and increased core fucosylation, while tamoxifen correlated with increased sialylation and decreased core fucosylation. These observations suggest potential immunomodulatory effects: anastrozole possibly reducing inflammation and tamoxifen impacting immune-mediated cytotoxicity. This study strongly emphasizes the importance of considering specific glycan traits to comprehend the dynamic mechanisms driving breast cancer progression and the effects of targeted therapies. The nuanced differences observed in glycan modifications between these two treatments underscore the necessity for further comprehensive research aimed at thoroughly evaluating the long-term implications and therapeutic efficacy for breast cancer patients. Full article

Through a comprehensive regional systematic collection, we conducted a genetic diversity analysis of wild red-fruited Lycium resources across the entire northwest region of China. This study provides a valuable genetic basis for germplasm exploration and the selection of new Lycium varieties. Utilizing fluorescence capillary electrophoresis, we carefully screened 16 pairs of SSR primers exhibiting high polymorphism. Subsequently, we inferred the genetic diversity of Lycium germplasm through structure clustering, UPGMA analysis, and molecular AMOVA. The 113 Lycium barbarum samples collected from northwest China exhibited distinct subgroups, namely the Qinghai–Gansu–Ningxia subgroup and the Xinjiang subgroup. These subgroups were clearly distinguishable based on genetic clustering. The genetic diversity within the samples was remarkably rich, as indicated by a mean I value of 1.04, He value of 0.57, and PIC value of 0.73. Notably, the majority of genetic diversity (72.99%) was found within populations, signifying substantial intrapopulation variation. Furthermore, our findings revealed significant genetic differentiation among populations, with a substantial Fst value of 0.27 and gene flow Nm estimated at 0.68. This suggests that the genetic variation levels in northwest Lycium were notably high, primarily driven by pronounced genetic differentiation among populations. Nonetheless, it is important to note that genetic diversity predominantly persists within populations. The observed subpopulation structure of Qinghai–Gansu–Ningxia and Xinjiang regions in northwest China can be primarily attributed to geographical isolation. These geographical barriers have played a pivotal role in shaping the genetic differentiation and structure of Lycium populations in the region. Consequently, our study sheds light on the complex genetic landscape of Northwest Lycium and highlights the significance of considering both within-population diversity and population differentiation in conservation and breeding programs. Full article

Capillary zone electrophoresis (CZE) is an important technique for the analysis of monoclonal antibodies (mAbs). A recently released light-emitting diode (LED)-induced fluorescence (LEDIF) detector equipped with a 275 nm LED for the detection of proteins through their native fluorescence was used in this study and compared to results obtained using the predominant detection mode, the measurement of the absorption of ultraviolet light (UV detection). This was accomplished using an established CZE method for the analysis of three mAbs: NISTmAb, matuzumab, and Intact Mass Check Standard (Waters). For this purpose, the detector’s settings were first optimized using a design of experiments approach. Three factors, rise time, photomultiplier high voltage supply, and acquisition frequency, were optimized by means of a D-optimal design. The optimal settings were then used for the investigation of signal-to-noise ratios (S/Ns), linearity, and precision. LEDIF detection offered a similar separation quality, up to 12 times higher S/Ns, and lower limits of detection compared to UV detection. Repeatability was excellent, with relative standard deviations (RSDs) of approximately 1% for percentage areas. For intermediate precision, RSDs of <2% (n = 3 × 10) were typically achieved. Overall, LEDIF detection was found to be an excellent and easily optimizable alternative to UV detection. Full article

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis–sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization. Full article