An enzyme-free and label-free multiplex detection of miRNAs by entropy-driven circuit coupled with capillary electrophoresis.

MicroRNAs (miRNAs) are currently recognized as important biomarkers for the early diagnosis and prognostic treatment of cancer. Herein, we developed a simple and label-free method for the multiplex detection of miRNAs, based on entropy-driven circuit (EDC) amplification and non-gel sieving capillary electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform. In this system, three different lengths of fuel chains were designed to catalyze three EDC, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. In the presence of target miRNA, the EDC cycle amplification reaction was triggered, generating numerous stable double-strands products (F-DNA/L-DNA). Since the three miRNAs correspond to three different lengths of F-DNA/L-DNA, they can be easily isolated and detected by NGCE. This strategy has good sensitivity, with detection limits of 68 amol, 292.2 amol, and 394 amol for miRNA-21, miRNA-155, and miRNA-10b, respectively. Additionally, this method has good specificity and can effectively distinguish single-base mismatches of miRNA. The recoveries of the three miRNAs in deproteinized healthy human serum ranged from 91.28 % to 108.4 %, with a relative standard deviation (RSD) of less than 7.9 %. This method was further applied to detect cellular miRNAs in human breast cancer (MCF-7) cell extracts, revealing an up-regulation of miRNA-21, miRNA-155, and miRNA-10b in MCF-7 cells. The successful spiked recovery in human serum and RNA extraction from MCF-7 cells underscores the practicality of this method. Therefore, this strategy has broad application prospects in biomedical research.

Speciation of selenium-containing small molecules in urine and cell lysate by CE-ICPMS with in-capillary enrichment.

The quantitative speciation of selenium in biological systems is highly important for evaluating health status and elucidating transformations of Se species in physiological and pathological processes. Hyphenation of capillary electrophoresis with inductively coupled plasma mass spectrometry (CE-ICPMS) is promising for this purpose. However, the unfavorable or insufficient sensitivity for selenium analysis with CE-ICPMS seriously limits its practical applications in biological analysis, e.g., cell analysis. Therefore, it is crucial to improve the detection sensitivity for Se species. In this study, CE-ICPMS sensitivities for five selenium species (selenocystamine (SeA), methyl-2-acetamido-2-deoxy-1-seleno-ß-d-galactopyranoside (SeSug 1), selenomethionine (SeMet), Se-Methylselenocysteine (MeSeCys) and selenocystine (SeCys)) were improved by in-capillary stacking via pH gradient between the zones of sample-leading buffer and the incorporation of isopropanol. The improvement on sensitivity of up to 9.9 folds was achieved in different biological samples, with LODs of 0.29-0.52 µg L-1. This approach was further applied for Se speciation in cell lysate, urine and culture medium. It showed that SeMet was more readily reduced in the medium and favorably accumulated by HepG2, HuH-7 and HCCLM3 cells with respect to SeSug 1 and MeSeCys. In cells, all the three Se species were largely transformed into other Se species. Furthermore, more than 70 % of SeMet reduced in medium was transformed into unknown Se species after 48-h interaction with cells.

Reducing the water quenching processes using heavy water in capillary electrophoresis with fluorescence detection.

Water, ubiquitous in analytical methods, is renowned for its fluorescence quenching properties, influencing techniques like fluorescence spectrophotometry or techniques with fluorescence detection. This study explores the impact of water (H2O) substitution for heavy water (D2O) on the fluorescence behavior of anthraquinones and anthracyclines. Anthraquinones and anthracyclines play crucial roles in pharmacy, serving as essential components in various therapeutic formulations, particularly in cancer treatment and other pharmacological interventions. Capillary electrophoresis (CE) with heavy water as the background electrolyte (BGE) solvent offers superior sensitivity to the separation and detection of these analytes. Experimental results demonstrate the improved detection limits and separation efficiency of selected anthraquinones rhein (RH), aloe-emodin (AE), and anthracyclines doxorubicin (DOX), epirubicin (EPI) and daunorubicine (DAU) in heavy water-based buffers, highlighting the potential of heavy water in advancing analytical chemistry.

Greening Separation and Purification of Proteins and Peptides.

The increasing awareness of environmental issues and the transition to green analytical chemistry (GAC) have gained popularity among academia and industry in recent years. One of the principles of GAC is the reduction and replacement of toxic solvents with more sustainable and environmentally friendly ones. This review gives an overview of the advances in applying green solvents as an alternative to the traditional organic solvents for peptide and protein purification and analysis by liquid chromatography (LC) and capillary electrophoresis (CE) methods. The feasibility of using greener LC and CE methods is demonstrated through several application examples; however, there is still plenty of room for new developments to fully realize their potential and to address existing challenges. Thanks to the tunable properties of designer solvents, such as ionic liquids and deep eutectic solvents, and almost infinite possible mixtures of components for their production, it is possible that some new designer solvents could potentially surpass the traditional harmful solvents in the future. Therefore, future research should focus mainly on developing new solvent combinations and enhancing analytical instruments to be able to effectively work with green solvents.

Profiling of metabolic dysregulation in ovarian cancer tissues and biofluids.

Ovarian cancer (OC) is the most lethal gynecologic cancer, mainly due to late diagnosis with widespread peritoneal spread at first presentation. We performed metabolomic analyses of ovarian and paired control tissues using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to understand its metabolomic dysregulation. Of the 130 quantified metabolites, 96 metabolites of glycometabolism, including glycolysis, tricarboxylic acid cycles, urea cycles, and one-carbon metabolites, showed significant differences between the samples. To evaluate the local and systemic metabolomic differences in OC, we also analyzed low or non-invasively available biofluids, including plasma, urine, and saliva collected from patients with OC and benign gynecological diseases. All biofluids and tissue samples showed consistently elevated concentrations of N1,N12-diacetylspermine compared to controls. Four metabolites, polyamines, and betaine, were significantly and consistently elevated in both plasma and tissue samples. These data indicate that plasma metabolic dysregulation, which the most reflected by those of OC tissues. Our metabolomic profiles contribute to our understanding of metabolomic abnormalities in OC and their effects on biofluids.

CE-UV method for the determination of catecholamine metabolites from baby pee-covered diapers.

A method has been developed for the analysis of vanillylmandelic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from baby urine as biomarkers of neuroblastoma in infants. Disposable diapers were employed as sampling devices in order to guarantee a low invasiveness during this step. The proposed method consists on a simple extraction step with water from the used diaper followed by the measurement using capillary electrophoresis with UV detection. The Box-Behnken design (BBD) was utilized to optimize the process of extracting catecholamine metabolites from the examined samples. The variables of the sample preparation step were optimized and the method was validated obtaining limits of quantification of 1.65 µg mL-1, good intraday and inter-day precision with RSDs under 15 %. Finally the method was applied to real samples collected from the Department of Neonatology, University Clinical Centre (Gdansk, Poland). The greenness of the proposed method was also evaluated with different tools (i.e., AGREEPrep and GAPI) with satisfactory results, which allow to state that the method can be considered green. Moreover, its practicality was evaluated by application of BAGI tool, proving to be a practical and economical method to be applied in routine laboratories for determination of catecholamine metabolites in urine-type samples.

Enhanced Commendable Sensitivity and Specificity for MSI in Colorectal Cancer by a New PCR-HRM Based 8-Loci MSI Assay.

BACKGROUND: This study evaluated the performance of the PCR-HRM assay by comparing it with immunohistochemistry (IHC) for mismatch repair (MMR) proteins and the PCR capillary electrophoresis (PCR-CE) methods. RESULTS: A total of 224 patients with colorectal cancer participated in the study, with nearly half having mismatch repair deficiency (dMMR) tissues and the remainder possessing pMMR tissues. There was a 97.77% concordance between the PCR-HRM assay and IHC, and a 97.56% concordance between PCR-HRM and the PCR-CE assay. In comparison with IHC for dMMR proteins, the PCR-HRM demonstrated a sensitivity of 96.36% and a specificity of 99.12%. When juxtaposed with the PCR-CE assay, its sensitivity was 98.96% and specificity stood at 96.33%. The mutations observed in the microsatellite loci were uniformly distributed across all eight loci. Discrepant outcomes were more frequent in instances of MLH1 and PMS2 deficiency. Furthermore, the germline mutation status of MLH1, MSH2, PMS2, and MSH6 in 62 patients was ascertained using next-generation sequencing. All patients displaying MMR gene pathogenic mutations (N = 14) were identified as MSI-H by PCR-HRM, whereas those with MSS tissues (N = 43) did not exhibit MMR gene pathogenic mutations. Thus, the PCR-HRM method proficiently pinpoints tumors with verified germline MMR mutations, indicative of Lynch syndrome. CONCLUSION: Conclusively, the PCR-HRM assay emerges as a swift and congruent diagnostic tool for microsatellite instability, boasting commendable sensitivity and specificity in colorectal cancer.

Capillary zone electrophoresis-tandem mass spectrometry for in-depth proteomics analysis via data-independent acquisition.

A capillary zone electrophoresis (CZE) system was coupled to an Orbitrap mass spectrometer operating in a data-independent acquisition (DIA) mode for in-depth proteomics analysis. The performance of this CZE-DIA-MS system was systemically evaluated and optimized under different operating conditions. The performance of the fully optimized CZE-DIA-MS system was subsequently compared to the one by using the same CZE-MS system operating in a data-dependent acquisition (DDA) mode. The experimental results show that the numbers of identified peptides and proteins acquired in the DIA mode are much higher than the ones acquired in the DDA mode, especially with the small sample loading amount. Specifically, the numbers of identified peptides and proteins acquired in the DIA mode are 1.8-fold and 2-fold higher than the ones acquired in the DDA mode by using 12.5 ng Hela digests. The proteins identified in the DIA mode also cover almost all the proteins identified in the DDA mode. In addition, a potential cancer biomarker protein, carbohydrate antigen 125, undetected in the DDA mode, can be easily identified in the DIA mode even with 12.5 ng Hela digests. The performance of the CZE-DIA-MS system for in-depth proteomics analysis with a limited sample amount has been fully demonstrated for the first time through this study.

Interference in HbA1c measurement: a case of electropherogram shift due to hyperleukocytosis leading to the discovery of leukemic mantle cell lymphoma.

HbA1c is a pivotal biomarker in diabetes management, reflecting long-term glycaemic control. HbA1c is often measured with capillary electrophoresis, which generally is a very precise technique, but there can be interference, especially in the case of haemoglobin diseases. Thus, in patients with underlying conditions, the accurate measurement of HbA1c can be challenging. We present a case of special interference in a 74-year-old female patient referred to a HbA1c test, in whom the measurement was thought to be significantly affected by hyperleukocytosis and led to an unexpected diagnosis of leukemic low-grade lymphoma. This case report highlights the underrecognized potential interference of leukocytosis in HbA1c measurement. It underscores the importance of clinical vigilance when interpreting HbA1c results in patients with underlying haematological conditions.

Comparison of multiplex PCR capillary electrophoresis assay and PCR-reverse dot blot assay for human papillomavirus DNA genotyping detection in cervical cancer tissue specimens.

Background: The study aimed to evaluate the positivity rates and genotype distribution of the multiplex PCR capillary electrophoresis (MPCE) and PCR-Reverse Dot Blot (PCR-RDB) assays for human papillomavirus (HPV) detection in cervical cancer tissue specimens, and to explore their detection principles and applications in large-scale population screening. Methods: The MPCE and PCR-RDB assays were performed separately on 425 diagnosed cervical cancer tissue specimens. Subsequently, the results of both assays were compared based on the HPV infection positivity rates and genotype distribution. Results: The overall positive rates of HPV genotypes for the MPCE and PCR-RDB assays were 97.9% and 92.9%, respectively. A p-value < 0.001 indicated a statistically significance difference in consistency between the two assays. The kappa value was 0.390, indicating that the consistency between both assays was fair. HPV16 was the most common single-genotype infection type, with infection rates detected via MPCE and PCR-RDB assays being 75.7% and 68.3%, respectively. In the age group >50 years, the HPV multiple-type infection rate detected via MPCE assay was significantly higher than that detected by the PCR-RDB assay, with a statistically significant difference (p = 0.002). Conclusion: To reduce the false-negative rate and improve screening efficiency, the MPCE assay, which targets the oncogenic gene E6/E7 segments, can be extended to the general female population for the early detection, diagnosis, and treatment of cervical cancer.

Determination of amino acids and peptides without their pre-column derivatization by capillary electrophoresis with ultraviolet and contactless conductivity detection. An overview.

This review provides an overview of recent works focusing on the determination of amino acids (AAs) and peptides using capillary electrophoresis with contactless conductivity detection and ultraviolet (UV) detection, which is the most widespread detection in capillary electromigration techniques, without pre-capillary derivatization. Available options for the UV detection of these analytes, such as indirect detection, complexation with transition metal ions, and in-capillary derivatization are described. Developments in the field of direct detection of UV-absorbing AAs and peptides as well as progress in chiral separation are described. A separate section is dedicated to using on-line sample preconcentration methods combined with capillary electrophoresis-UV.

Capillary Electrophoresis-Laser Induced Fluorescence Method Development and Validation for Quantification of Nine Gangliosides-Application to Analysis of Cell Lines of CNS Origin.

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.

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells.

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

[Highly sensitive detection of fluorescent whitening agents in flour using sheathless capillary electrophoresis-electrospray ionization-tandem mass spectrometry].

Fluorescent whitening agents (FWAs) are dyes that emit visible blue or blue-purple fluorescence upon ultraviolet-light absorption. Taking advantage of light complementarity, FWAs can compensate for the yellow color of many substances to achieve a whitening effect; thus, they are used extensively in various applications. FWAs are generally stable, but their presence in the environment can lead to pollution and accumulation in the body through the food chain. Recent studies have revealed that some types of FWAs, such as coumarin-based FWAs, may exhibit photo-induced mutagenic effects that can trigger allergic reactions in humans and even pose carcinogenic risks. Hence, the development of an accurate and highly sensitive method for detecting FWAs in food-related samples is a crucial endeavor. Owing to the high polarity and structural similarity of FWAs, the accurate determination of these substances in complex food samples requires an analytical method that offers both efficient separation and sensitive detection. Capillary electrophoresis (CE) exhibits essential features such as high separation efficiency, short analysis times, very small sample injection requirements, minimal use of organic solvents, and simple operation. Thus, it is often used as an effective alternative to liquid chromatographic techniques. Over the past few decades, electrospray ionization mass spectrometry (ESI-MS) has been utilized as a highly sensitive and accurate detection method in numerous chemical analytical fields because it enables the analysis of molecular structures. By combining the high separation efficiency of CE with the high sensitivity of ESI-MS, a powerful tool for identifying and quantifying trace amounts of FWAs in food samples may be obtained. In this study, we present a method based on sheathless CE coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the simultaneous detection of six trace FWAs in flour. In the proposed method, the CE separation device is directly coupled to the mass spectrometer through a sheathless interface without the need for a sheath liquid for electric contact, thereby avoiding the dilution of the analytes and improving detection sensitivity. Various conditions that could affect extraction recovery, separation efficiency, and detection sensitivity were evaluated and optimized. The FWAs were effectively extracted from the sample matrix with reduced matrix effects by ultrasonic-assisted extraction at a temperature of 30 ℃ for 20 min using CHCl3-MeOH (3∶2, v/v) as the extraction solvent. The extract was centrifuged, dried under N2, and reconstituted in CHCl3-MeOH (1∶4, v/v) for subsequent analysis. During the detection process, the CE device was coupled to the ESI-MS/MS instrument via a highly sensitive porous spray needle, which served as the sheathless electrospray interface. The target FWAs were scanned in positive-ion mode (ESI+) to ensure the stability and intensity of the obtained signals. Additionally, multiple-reaction monitoring (MRM) mode and MS/MS analysis were used to simultaneously quantify the six targets with high selectivity. The developed sheathless CE-ESI-MS/MS method detected the FWAs with high sensitivity over wide linear ranges with low method limits of detection (0.04-0.67 ng/g). The recoveries of the six target FWAs at three spiked levels were between 77.5% and 97.2%, with good interday (RSD≤11.5%) and intraday (RSD≤10.2%) precision. Analyses of the six target FWAs in eight commercial flour samples were performed using this method, and four positive samples were identified. These results demonstrate that the proposed CE-ESI-MS/MS method is a promising strategy for the determination of trace FWAs in complex food sample matrices with efficient separation and high sensitivity.

Development of capillary electrophoresis methods for the detection of microbial metabolites on potential future spaceflight missions.

The search for chemical indicators of life is a fundamental component of potential future spaceflight missions to ocean worlds. Capillary electrophoresis (CE) is a useful separation method for the determination of the small organic molecules, such as amino acids and nucleobases, that could be used to help determine whether or not life is present in a sample collected during such missions. CE is under development for spaceflight applications using multiple detection systems, such as laser induced fluorescence (LIF) and mass spectrometry (MS). Here we report CE-based methods for separation and detection of major polar metabolites in cells, such as amino acids, nucleobases/sides, and oxidized and reduced glutathione using detectors that are less expensive alternatives to LIF and MS. Direct UV detection, indirect UV detection, and capacitvely coupled contactless conductivity detection (C4D) were tested with CE, and a combination of direct UV and C4D allowed the detection of the widest variety of metabolites. The optimized method was used to profile metabolites found in samples of Escherichia coli and Pseudoalteromonas haloplanktis and showed distinct differences between the species.

Rational Design of Highly Selective Sialyllactose-Imprinted Nanogels.

We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE), to rapidly assess an affinity ranking. Finally, the best monomer 2-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant≈106 M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.

Metabolome analyses of skin dialysate: Insights into skin interstitial fluid biomarkers.

BACKGROUND: Metabolites in biofluids can serve as biomarkers for diagnosing diseases and monitoring body conditions. Among the available biofluids, interstitial fluid (ISF) in the skin has garnered considerable attention owing to its advantages, which include inability to clot, easy access to the skin, and possibility of incorporating wearable devices. However, the scientific understanding of skin ISF composition is limited. OBJECTIVE: In this study, we aimed to compare metabolites between skin dialysate containing metabolites from the skin ISF and venous blood (plasma) samples, both collected under resting states. METHODS: We collected forearm skin dialysate using intradermal microdialysis alongside venous blood (plasma) samples from 12 healthy young adults. We analyzed these samples using capillary electrophoresis-fourier transform mass spectrometry-based metabolomics (CE-FTMS). RESULTS: Significant positive correlations were observed in 39 metabolites between the skin dialysate and plasma, including creatine (a mitochondrial disease biomarker), 1-methyladenosine (an early detection of cancer biomarker), and trimethylamine N-oxide (a posterior predictor of heart failure biomarker). Based on the Human Metabolome Technologies database, we identified 12 metabolites unique to forearm skin dialysate including nucleic acids, benzoate acids, fatty acids, amino acids, ascorbic acid, 3-methoxy-4-hydroxyphenylethyleneglycol (an Alzheimer's disease biomarker), and cysteic acid (an acute myocardial infarction biomarker). CONCLUSION: We show that some venous blood biomarkers may be predicted from skin dialysate or skin ISF, and that these fluids may serve as diagnostic and monitoring tools for health and clinical conditions.

Predictive analysis of breast cancer response to neoadjuvant chemotherapy through plasma metabolomics.

PURPOSE: Preoperative chemotherapy is a critical component of breast cancer management, yet its effectiveness is not uniform. Moreover, the adverse effects associated with chemotherapy necessitate the identification of a patient subgroup that would derive the maximum benefit from this treatment. This study aimed to establish a method for predicting the response to neoadjuvant chemotherapy in breast cancer patients utilizing a metabolomic approach. METHODS: Plasma samples were obtained from 87 breast cancer patients undergoing neoadjuvant chemotherapy at our facility, collected both before the commencement of the treatment and before the second treatment cycle. Metabolite analysis was conducted using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry (LC-MS). We performed comparative profiling of metabolite concentrations by assessing the metabolite profiles of patients who achieved a pathological complete response (pCR) against those who did not, both in initial and subsequent treatment cycles. RESULTS: Significant variances were observed in the metabolite profiles between pCR and non-pCR cases, both at the onset of preoperative chemotherapy and before the second cycle. Noteworthy distinctions were also evident between the metabolite profiles from the initial and the second neoadjuvant chemotherapy courses. Furthermore, metabolite profiles exhibited variations associated with intrinsic subtypes at all assessed time points. CONCLUSION: The application of plasma metabolomics, utilizing CE-MS and LC-MS, may serve as a tool for predicting the efficacy of neoadjuvant chemotherapy in breast cancer in the future after all necessary validations have been completed.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.