A compact visible-light spectrometric detector for capillary electrophoresis.

The purity of commercial cucumber varieties, predominantly F1 hybrids, is a critical factor in seed quality assessment. While SSR or SNP markers are commonly used, insertion-deletion (InDel) markers offer greater stability and practical advantages, including lower cost, compatibility with agarose gel electrophoresis, and suitability for multiplex PCR. In this study, we developed long InDel (> 30bp) markers for variety purity detection in cucumber using whole-genome resequencing data from 182 accessions. A total of 326,479 InDel loci were identified, of which 2655 long InDels satisfy the following requirements which have conserved 200bp flanking sequences, high polymorphism and suitability for primer design. From these, 74 markers with minor allele frequency (MAF) > 0.2 across four cucumber populations were evenly distributed across seven chromosomes and synthesized. All markers successfully amplified target products, and 10 exhibited high heterozygosity and clear bands on agarose gels. These 10 markers were further adapted for multiplex capillary electrophoresis detection by adding different fluorescent groups. DNA fingerprints of 66 commercial cucumber varieties were constructed using these markers. Among them, four markers InD_Cu35, InD_Cu46, InD_Cu61 and InD_Cu66, showed at least one heterozygous genotype across all varieties, making them particularly suitable for purity testing of cucumber hybrid varieties. The practical utility of these markers was confirmed by assessing the purity of a new hybrid variety, Lvlinglong (98.9%). These long InDel markers provide a robust, flexible, and cost-effective tool for routine cucumber variety purity detection and genetic analysis.

Forensic microbiology allows identification by microbiome analysis when human DNA analysis is limited. However, microbiome-based approaches have not been widely used in forensic medicine. The spatial and temporal stability of microbial communities in response to environmental exposures represents a serious challenge. This study investigates the dynamics of skin and saliva microbial markers in response to environmental exposure to advance their forensic applicability. We selected 8 characteristic microorganisms (Finegoldia magna, Corynebacterium tuberculostearicum, Cutibacterium acnes for skin; Haemophilus parainfluenzae, Streptococcus oralis, Prevotella melaninogenica for saliva; Achromobacter, Pseudomonas as environmental markers) and developed two multiplex amplification systems for capillary electrophoresis detection. A longitudinal exposure model (0-120 days) under controlled indoor and dry environment was applied to 420 skin and saliva samples from 10 subjects. Saliva samples exhibited significant microbial community shifts between environments (R2 = 0.4137). Dry environment preserved community structure better, evidenced by delayed inflection points in markers' proportions and stronger clustering in PCoA. Redundancy analysis correlated microbial markers with temperature and humidity, and this correlation varied. The microbial markers remained detectable after 120 days of environmental exposure and were able to provide information on community characteristics and deposition time. This study explored the effects of environment and time on microbial communities and the response patterns of microbial markers to temperature and humidity. It is expected to provide help for forensic microbial research related to environmental exposure.

Advances in the detection of azodicarbonamide and the metabolic product semicarbazide.

Molecularly imprinted polymers based enrichment and separation for trace analysis in capillary electrophoresis.

Analysis of lysozyme in human saliva by CE-UV: A new simple, fast, and reliable method.

Homochiral zeolite imidazolateframework-8 (ZIF-8) combines the inherent characteristics of ZIF-8 with the chiral recognition function and open chiral channel, becoming one preferred choice for enantiomer separation. In this work, ZIF-8 with D-histidine as the chiral carbon center (D-His-ZIF-8) was introduced into the pores to enhance the extraction performance of hollow-fiber liquid-phase microextraction (HF-LPME). A novel method was developed for the selective and sensitive determination of chiral amino acids related to the nervous system by D-His-ZIF-8@HF-LPME coupled with capillary electrophoresis and laser-induced fluorescence detection. Under the optimal conditions, D-type amino acids could obtain higher enrichment efficiency than L-type ones due to the stronger geometry-dependent hydrogen-bonding interactions between L-type amino acids and the chiral channels of ZIF-8 materials modified with D-His, and the maximum enrichment factor reached 640 times (for D-serine). This developed method has been successfully applied to the analyses of four chiral amino acids (D/L-serine and D/L-aspartic acid) in exhaled breath condensate and saliva samples of healthy volunteers. The limits of detection were 0.030-0.30nM (S/N = 3) in the real sample matrixes. This developed method meets the principles of green analytical chemistry and achieves a greenness score of 80 assessed by the analytical eco-scale tool. This proposed method provides one green alternative for the sensitive and accurate analyses of chiral amino acids in the non-invasive body fluids.

Separations of enantiomers by capillary electrophoresis (CE) are achieved upon selective interaction of each enantiomer with a chiral selector, resulting in the formation of supramolecular aggregates, or complexes, that may reach the CE detector at different velocities. In order to understand (rationalize) the enantioseparation process and also to be able to modulate its outcome, it is necessary to discern the structure and the stability of these supramolecular complexes. Nuclear magnetic resonance (NMR) constitutes a powerful instrument to elucidate the structure of the complexes at the molecular level. A very important feature of NMR is that the samples to be analyzed can be prepared in a way that mimics the experimental conditions employed in the CE runs, like the pH and composition of the CE conducting medium and the operational temperature.This tutorial describes in detail the necessary steps, from beginning to end, to conduct those NMR experiments to determine the stoichiometry, the values of the association constants, and the 3D structure of the complexes. Precise practical instructions on how to prepare NMR samples from deuterated buffers and stock solutions are given. Other factors influencing the outcome of the NMR analyses, like acquisition parameters of NMR experimental protocols, are also discussed. This chapter pretends to be a concise guide with which researchers with a basic knowledge of NMR can carry out a general and elaborate study into CE-mediated enantioseparations. The stoichiometry and the binding constants of the complexes are determined by titration experiments, whereas 3D structures of the complexes are built upon spotting intermolecular interactions between hydrogen atoms of the analyte and the chiral selector.

Fumarate, succinate, maleate, dihydroxyfumarate, D–tartarate, L–tartarate, DL–tartarate, L-malate, D-malate, oxaloacetate, citrate, and DL-isocitrate in the 5–100 μM concentration range were incubated in 12.5 mM HEPES/25 mM TRIS base containing 200 μM Eu3+–tetracycline and 60% (v/v) formamide (pH unadjusted). After 30 min of incubation, they were separated at 4 °C by capillary electrophoresis utilizing laser-induced luminescence detection with 12.5 mM HEPES/25 mM TRIS base containing 60% formamide as the running buffer. All analytes yielded peaks, with the exception of fumarate, succinate, and maleate. L-Malate was detected down to 100 nM. The main component of this study was the analysis of malate. The objective was to develop a stereoselective methodology for the detection of L-malate. This was achieved by varying the formamide concentration and separation temperature. When the temperature was increased to 22 °C and the formamide concentration decreased to 40%, the sensitivity for L-malate was diminished about 10-fold, but that for D-malate was eliminated. This combination of conditions allowed for the stereospecific analysis of L-malate.

High sensitivity and specificity analysis of multiple miRNAs.

In China, the traditional method for analyzing soil available phosphorus is inadequate for large-scale soil assessment and nationwide soil formulation demands. To address this, we propose a rapid and reliable method for soil-available phosphorus detection. The setup includes an on-site rapid pre-treatment device, a non-contact conductivity detection device, and a capillary electrophoresis buffer solution system composed of glacial acetic acid and hydroxypropyl-β-cyclodextrin. The on-site rapid pre-treatment process includes fresh soil moisture content detection (moisture rapid detector), weighing (handheld weighing meter), stirring (handheld rapid stirrer), and filtration (soil rapid filter) to obtain the liquid sample, and direct injection (capillary electrophoresis detector). The phosphate ion detection parameters include capillary size, separation voltage, injection parameters, and electric injection. We used Liaoning brown soil, Henan yellow tidal soil, Heilongjiang black soil, and Anhui tidal soil as standard samples. Additionally, we used mathematical modeling methods and machine learning algorithms to analyze and process research data. Following calibration with standard samples, the experimental blind test samples demonstrated conformity with the national standard method, exhibiting a relative standard deviation of less than 3%. The proposed pre-treatment device and non-contact conductivity detector are powered by lithium-ion batteries, rendering them ideal for extended field operations. The non-contact conductivity detector obviates the need for direct contact with test samples, mitigating environmental pollution. Furthermore, the neural network model exhibited the highest level of goodness of fit in chemical data analysis.

Biarsenical-based fluorescent labeling of metallothioneins as a method for ultrasensitive quantification of poly-Cys targets.

This paper introduces a novel contactless single-chip detector that utilizes impedance-to-digital conversion technology to measure impedance in the microfluidic channel or capillary format analytical device. The detector is designed to operate similarly to capacitively coupled contactless conductivity detectors for capillary electrophoresis or chromatography but with the added capability of performing frequency sweeps up to 200 kHz. At each recorded data point, impedance and phase-shift data can be extracted, which can be used to generate impedance versus frequency plots, or phase-shift versus frequency plots. Real and imaginary parts can also be calculated from the data, allowing for the generation of Nyquist diagrams. This detector represents the first of its kind in the contactless conductivity class to provide spectrum-type data, as demonstrated in capillary electrophoresis experiments.

Quality control of fish sauce by simultaneous determination of histamine and tyramine with capillary electrophoresis and contactless conductivity detection

Background In liquid biopsy, mutation detection is primarily performed using cell-free DNA (cfDNA). However, the numerous advantages of extracellular vesicle (EV) DNA for mutation detection have gradually garnered the attention of researchers in recent years. This study aimed to compare the differences between EV DNA and cfDNA in mutation detection and explore the role of plasma androgen receptor (AR) mutations in the prognosis of prostate cancer (PCa). Methods We compared the biological characteristics of plasma extracellular vesicle DNA (p-EV DNA) and cfDNA by capillary electrophoresis and concentration detection. Subsequently, we performed pan-oncogene-targeted sequencing in paired tissue and plasma samples from five patients with PCa to verify the feasibility of mutation detection using p-EV DNA and cfDNA. Further, we conducted AR mutation detection in expanded samples to compare the differences between EV DNA and cfDNA in mutation detection and to analyse their role in PCa. Results p-EV DNA fragments were larger than plasma cell-free DNA (p-cfDNA) fragments; however, there was no significant difference in their concentrations in the plasma of patients with PCa. Feasibility analysis revealed that major mutations associated with PCa detected in tissue samples could be identified in both p-EV DNA and p-cfDNA. Advantage comparison found that, although cfDNA could detect more mutations, AR mutations in EV DNA were more strongly associated with a poor prognosis of PCa than cfDNA. Conclusion Mutation detection using either EV DNA or cfDNA is both feasible in PCa liquid biopsies, and EV DNA AR mutations have an advantage in prognostic assessment for PCa. This study lays the foundation for future research on EV DNA-related biomarkers.

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.

Future life-detection missions will likely search for biosignatures within a wide range of organic compounds, including fatty acids. In order to determine such biosignatures, it is necessary to identify and quantify individual fatty acids present within a sample. In this study, we present a method using capillary electrophoresis coupled to contactless conductivity detection (CE-C4D) for the separation and detection of both saturated and unsaturated fatty acids after derivatization with N,N-diethylethylenediamine, triethylamine, and 2-chloro-1-methylpyridinium iodide at 40°C for 10min. Operating conditions (background electrolyte, separation voltage, and temperature) were optimized to provide maximum separation of fatty acids, thereby allowing their identification and quantification. Using a background electrolyte of 2M acetic acid in 45% acetonitrile, an optimal separation was obtained with a separation voltage of 10kV and a capillary temperature of 15°C. The optimized CE-C4D method was used to analyze samples of the cyanobacterium Spirulina. Multiple fatty acids were detected in the samples, showing the potential of this method for detection of fatty acid biosignatures during future spaceflight missions.

A simple method to remove polyimide coating from fused silica capillaries for capillary electrophoresis

Peptide substrate reporters are short chains of amino acids designed to act as substrates for enzymes of interest. Combined with capillary electrophoresis and laser-induced fluorescence detection (CE-LIF), they are powerful molecular tools for quantitative measurements of enzyme activity even at the level of single cells. Although most peptide substrate reporters have been optimized for human or murine cells in health-related applications, their performance in nonmammalian organisms remains largely unexplored. In this study, we evaluated three peptide substrate reporters for protein kinase B (PKB) in two eukaryotic microbes, Dictyostelium discoideum and Tetrahymena thermophila, which are evolutionarily distant from mammals and from each other yet express PKB homologues. All three peptide substrate reporters were phosphorylated in lysates from both organisms but with varying phosphorylation kinetics and stability. To demonstrate reporter utility, we used one to screen for and identify the previously unknown stimulus needed to activate PHK5, the PKB homologue in T. thermophila. In D. discoideum, we employed the highly quantitative nature of these assays using CE-LIF to make precise measurements of PKB activity in response to transient stimulation, drug treatment, and genetic mutation. These results underscore the broad applicability of peptide substrate reporters across diverse species while highlighting the need for further research to determine effective peptide stabilization strategies across different biological contexts.

Refractive index (RI) detection using backscatter interferometry (BSI) enables universal detection in capillary electrophoresis (CE). BSI detection is a versatile on-capillary approach that is easily integrated with capillary or microfluidic channels, straightforward to miniaturize, and inexpensive. The focused BSI light source can also double as the excitation source for fluorescence, enabling simultaneous universal (BSI) and specific (fluorescence) signals from the same detection volume. To improve BSI detection and expand orthogonal content, we integrate photothermal absorption with BSI detection. Nonradiative relaxation of an excited analyte releases heat into the surroundings, which modifies both the local RI and conductivity (viscosity) of the analyte zone. We recently showed that the BSI signal is sensitive to both RI and conductivity, which makes photothermal absorption a promising route to signal enhancement. Here, we use coaxially delivered BSI and photothermal absorption beams to characterize BSI, photothermal BSI, and fluorescence detection using the separation of test samples. We show that photothermal absorption leads to 3 orders of magnitude improvement in BSI detection limits at the powers studied and provides new opportunities for studying binding interactions with CE.