High-throughput and high-sensitivity capillary electrophoresis–mass spectrometry method for sulfur-containing amino acids

Biological thiol amino acids have been suggested as biomarkers for pathological changes because they are reactive chemicals that participate in various physiological processes. In this study, multisegmented injection capillary electrophoresis–mass spectrometry with online sample preconcentration was used for analysis of thiol amino acids and intermediates of sulfur metabolism in human glioma cell line U-251 with high accuracy, throughput, and sensitivity. This was achieved using multiple, large-volume injections for online sample preconcentration. The 16 intermediates of sulfur metabolism had a good linear correlation coefficient range of 0.984–1 and the limit of detection range was 1.4–203.9 ng/mL. The recovery ranges of most amino acids were 88.1–114.5%, 89.0–104.3%, and 76.9–104.5% at 0.3, 0.75, and 1.5 μg/mL, respectively. The relative standard deviation ranges for the inter- and intra-day precision were 1.8–10.7% and 4.3–18.8%, respectively. Compared with the traditional injection method, the analytical time for compounds in sulfur metabolism was reduced to 4 min/sample, the method throughput was enhanced five times, and the sensitivity was increased 14.4–33.1 times. Customized injection sequences were applied in experimental optimization. The developed method simplified the experimental optimization to one injection and is suitable for the analysis of sulfur metabolites in biological samples and has high sensitivity, throughput, speed, and accuracy.

Determination of 13-cis-Retinoic Acid and Its Metabolites in Plasma by Micellar Electrokinetic Chromatography Using Cyclodextrin-Assisted Sweeping for Sample Preconcentration

The online preconcentration technique, cyclodextrin-assisted sweeping (CD-sweeping), coupled with micellar electrokinetic chromatography (MEKC) was established to determine 13-cis-retinoic acid (13-cis-RA), all-trans-retinoic acid (all-trans-RA) and 4-oxo-13-cis-retinoic acid (4-oxo-13-cis-RA) in human plasma. A CD-sweeping buffer (45 mM borate (pH 9.2), containing 80 mM sodium dodecyl sulfate (SDS) and 22 mM hydroxypropyl β-CD (HP-β-CD) was introduced into the capillary and, then, the sample dissolved in 70 mM borate (pH 9.2): methanol = 9:1 (v/v) was injected into capillary by pressure. The separation voltage was 23 kV. Compared to the conventional cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method, the new technique achieved 224–257-fold sensitivity enrichment of analytes. The limits of detection of 13-cis-RA, all-trans-RA were 1 ng/mL, whereas that of 4-oxo-13-cis-RA was 25 ng/mL in plasma. The linear ranges of 13-cis-RA, all-trans-RA were between 15 and 1000 ng/mL, whereas that of 4-oxo-13-cis-RA was between 75 and 1500 ng/mL. The coefficient of correlation between the concentration of analytes and peak area ratio of analytes and internal standard (2, 4-dihydroxy-benzophenone) for intra-day (n = 3) and inter-day (n = 5) analyses were both greater than 0.999. The optimized experimental conditions were successfully applied to determine 13-cis-retinoic acid and its metabolites in plasma samples from a patient during the administration of 13-cis-RA for treating acne.

Recent Advances in Microfluidic Devices for Contamination Detection and Quality Inspection of Milk

Milk is a necessity for human life. However, it is susceptible to contamination and adulteration. Microfluidic analysis devices have attracted significant attention for the high-throughput quality inspection and contaminant analysis of milk samples in recent years. This review describes the major proposals presented in the literature for the pretreatment, contaminant detection, and quality inspection of milk samples using microfluidic lab-on-a-chip and lab-on-paper platforms in the past five years. The review focuses on the sample separation, sample extraction, and sample preconcentration/amplification steps of the pretreatment process and the determination of aflatoxins, antibiotics, drugs, melamine, and foodborne pathogens in the detection process. Recent proposals for the general quality inspection of milk samples, including the viscosity and presence of adulteration, are also discussed. The review concludes with a brief perspective on the challenges facing the future development of microfluidic devices for the analysis of milk samples in the coming years.

Origami paper-based sample preconcentration using sequentially driven ion concentration polarization

To overcome the hurdles of the ICP-based preconcentrator (i.e., instability and low efficiency) under physiological conditions, we proposed a novel approach by using a sequentially driven ICP process, showing a 13-fold preconcentration factor (PF) in human serum.

Polymeric Magnetic Microparticles as Electrochemical Immunosensing Platforms

Magnetic microparticles (MMPs) have been notably used as platforms in biosensing. Due to their magnetic behavior, they simplify purification and separation procedures, reducing time of analysis. They also allow sample preconcentration, minimizing matrix effects, which is of key relevance for applications using real samples. Even though there is a great number of commercially available MMPs, their performance is not always reliable. In this work, we propose the synthesis of novel polymeric MMPs for their use as electrochemical immunosensing platforms. Initially, magnetic nanoparticles of a diameter of 12 ± 2 nm and a saturation magnetization of 70 emu/g were synthesized and characterized. Then, they were encapsulated in a polymeric matrix of poly (lactic-co-glycolic) acid (PLGA), generating MMPs with a diameter of 90 ± 18 nm. Later, MMPs were functionalized with polyethyleneimine (PEI) as an intermediate step for the immobilization of affinity proteins or antibodies, necessary in electrochemical immunosensing. This would allow the obtaining of MMPs comparable to the commercially available ones but possessing higher saturation magnetization. The use of such MMPs could facilitate the detection of analytes of interest in diagnostics, among other applications.

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis

Here we present the result of our work on achieving sub-pM limit of quantitation in direct quantitative analysis of multiple miRNAs by capillary electophoresis with laser-induced fluorescence detection. The PDF file contains the description of the results with figures, references, and supporting information. The <i>raw data.ZIP</i> file is the archive containing (i) Excel files of raw data used to build figures presented in the PDF file and (ii) the <i>red-me.PDF </i>file explaining how to read and use the Excel files. The <i>figure data.ZIP</i> file is the archive of all figure files in Origin and Adobe Illustrator formats. <div><b>Abstract:</b> Thousands of putative miRNA-based cancer biomarkers have been reported but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method which is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. Capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard) but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here we explain challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly-negatively charged PNA–miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA–miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers. <div><br></div></div>

Necessity and Challenges of Sample Preconcentration in Analysis of Multiple MicroRNAs by Capillary Electrophoresis

Here we present the result of our work on achieving sub-pM limit of quantitation in direct quantitative analysis of multiple miRNAs by capillary electophoresis with laser-induced fluorescence detection. The PDF file contains the description of the results with figures, references, and supporting information. The <i>raw data.ZIP</i> file is the archive containing (i) Excel files of raw data used to build figures presented in the PDF file and (ii) the <i>red-me.PDF </i>file explaining how to read and use the Excel files. The <i>figure data.ZIP</i> file is the archive of all figure files in Origin and Adobe Illustrator formats. <div><b>Abstract:</b> Thousands of putative miRNA-based cancer biomarkers have been reported but none has been validated for approval by the Food and Drug Administration. One of the reasons for this alarming discrepancy is the lack of a method which is sufficiently robust for carrying out validation studies, which may require analysis of samples from hundreds of patients across multiple institutions and pooling the results together. Capillary electrophoresis (CE)-based hybridization assay proved to be more robust than reversed transcription polymerase chain reaction (the current standard) but its limit of quantification (LOQ) exceeds 10 pM while miRNA concentrations in cell lysates are below 1 pM. Thus, CE-based separation must be preceded by on-column sample preconcentration. Here we explain challenges of sample preconcentration for CE-based miRNA analyses and introduce a preconcentration method that can suit CE-based miRNA analysis utilizing peptide nucleic acid (PNA) hybridization probes. The method combines field-amplified sample stacking (FASS) with isotachophoresis (ITP). We proved that FASS-ITP could retain and concentrate both near-neutral PNA with highly-negatively charged PNA–miRNA hybrids. We demonstrated that preconcentration by FASS-ITP could be combined with the CE-based separation of the unreacted PNA probes from the PNA–miRNA hybrids and facilitate improvement in LOQ by a factor of 140, down to 0.1 pM. Finally, we applied FASS-ITP-CE for simultaneous detection of two miRNAs in crude cell lysates and proved that the method was robust when used in complex biological matrices. The 140-fold improvement in LOQ and the robustness to biological matrices will significantly expand the applicability of CE-based miRNA analysis, bringing it closer to becoming a practical tool for validation of miRNA biomarkers. <div><br></div></div>