ELECTROPHORESIS最新文献

Non-invasive prenatal paternity testing (NIPPT) enables the genotyping of cell-free DNA (cfDNA) from maternal plasma through deep sequencing. Microhaplotypes (MHs) combine the advantages of short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs) and have attracted much attention in NIPPT. In this study, we optimized 45 MHs from our previous study and confirmed the effectiveness of the 45plex MH panel through different kinship inferences using real samples, including duos, trios, full siblings, and second-to-fifth-degree relatives, and excluding unrelated individuals. Furthermore, we tested 11 cfDNA and reference mother–child pairs in the first trimester (7 + 4–12 + 6 weeks) and 11 cfDNA and reference trios in the second trimester (18+ weeks). The R packages Familias and RelMix and the software EuroForMix were used for data interpretation. The results showed that MHs of cfDNA could be effectively detected using our sequencing and genotyping pipelines. We correctly determined paternity in 11 NIPPT cases, with Log₁₀LR > 10, which were significantly separated from real unrelated males. Our study indicates that this massively parallel sequencing (MPS)-based 45plex MH panel provides more robust relationship inference capabilities than standard STR systems, complements NIPPT, and may help solve relevant issues for relative DNA mixtures.

A simple and inexpensive method for integrating universal refractive index (RI) detection with existing capillary electrophoresis (CE) platforms utilizing epifluorescence detection is demonstrated. The approach uses the same epi-illumination laser source used for fluorescence detection to create a forward-scattered interference pattern for RI detection. The forward-scattered interferometry (FSI) approach only requires the addition of a bicell photodiode detector and mechanism for aligning it in the fringe pattern to add complementary universal RI detection with existing highly specific epi-fluorescence detection. FSI and fluorescence electropherograms illustrate the utility of measuring both channels simultaneously. Because both signals are generated from the same laser source focused into the same detection zone, the electropherograms are perfectly registered. The sample plug is directly detected with FSI in every separation, providing an intrinsic marker of the electroosmotic flow (EOF) without the need for neutral markers or separate measurements. Moreover, continuous EOF monitoring is also demonstrated using the introduction of thermal markers that are sensed in the FSI signal. Finally, it is shown that the FSI signal responds to both RI and the separation voltage, as has been shown previously for back-scatter interferometry (BSI) detection. This leads to enhanced signal-to-noise at elevated separation voltages, conditions that reduce analysis time and improve peak efficiency.

In dairy products, Bacillus subtilis (B. subtilis) is considered a harmful spoilage bacterium. Consequently, it is imperative to establish highly sensitive and selective approaches for detecting B. subtilis. In this article, quantification of B. subtilis via its 16S rRNA was first performed by microchip electrophoresis (MCE) combined with a dual nucleic acid recycling amplification strategy involving apyrimidinic endonuclease 1 (APE1)-mediated cycle and catalytic hairpin assembly (CHA). In APE1-mediated cycle, two specially designed probes containing apurinic/apyrimidinic sites (AP sites) capture B. subtilis 16S rRNA, and then APE1 cleaves the AP sites to release the recycled target RNA. Next, the product of APE1-mediated cycle triggers the CHA and generates the final product for further MCE detection. Under the optimal conditions, the limit of detection for B. subtilis was 29 CFU/mL (S/N = 3). The proposed strategy was successfully applied to the detection of B. subtilis in pasteurized milk and exhibited high speed, high sensitivity, and good specificity.

A novel post-modification strategy was developed for rapid functionalization of monoliths through amino-yne click chemistry. This approach enabled the conjugation of activated alkynes onto amino-functionalized organic-silica hybrid monolith surfaces under mild, catalyst-free conditions. Systematic investigation of critical reaction parameters was conducted to optimize the post-modification process. The morphological and structural characteristics of the prepared monolith were characterized by scanning electron microscopy (SEM) and nitrogen adsorption measurements. The successful grafting of functional groups onto the monolith surface was confirmed by contact angle analysis, Fourier transform infrared (FT-IR) spectroscopy, and x-ray photoelectron spectroscopy (XPS). The functionalized monolith demonstrated chromatographic selectivity for diverse analytes, including phenolic compounds and some weakly polar/nonpolar compounds (benzoin/benzoin methyl ether, styrene/p-chlorostyrene, and 1-naphthylethylamine/naphthalene). Furthermore, it was successfully applied to the quantitative analysis of honokiol in authentic magnolol samples, showcasing its potential for practical analytical applications.

Electric droplet sorting is widely applied in the screening of target molecules, cells, drugs, and microparticles. Previous studies have made several optimizations on the electrode materials, structures, and arrangements. However, voltages of over 1 kV are required to realize droplet sorting, which causes the undesired droplet splitting. In addition, great droplet deformation caused by the high voltage decreases the sorting accuracy and generates negative effects on the targets encapsulated within the droplets. In this study, we developed a low-voltage–driven droplet sorter (LV-DS) for droplet sorting with high stability and small deformation. Our LV-DS consisted of one square active and four triangular ground electrodes which were fabricated using liquid metal with higher stability and higher electrical conductivity than the previous electrode materials of low-melting alloy and salt water. Our LV-DS could realize the high-stability droplet sorting with small deformation at the voltage as low as 80 V. The results showed that our LV-DS presented great prospects in the high-stability droplet sorting with a low voltage. In addition, the advantage of small droplet deformation makes our LV-DS be attractive for the applications, such as droplet-based cell sorting with a high viability and droplet-based material screening with a small deformation.

Foodborne pathogenic bacteria always threaten human health. Flavonoids are commonly used in antibacterial applications. Studying the antibacterial effect of flavonoids on bacteria is significant. Capillary electrophoresis is a versatile separation means. It has different separation modes, so, it can be applied in the analysis of both bacteria and flavonoids. In this study, an efficient method of indirect separation and detection of three bacteria Vibrio parahaemolyticus, Escherichia coli, and Staphylococcus aureus was developed. The determination of bacteria was realized by separating the aptamers corresponding to the bacteria by capillary sieving electrophoresis. This method avoided the disadvantages of low resolution, channel adsorption and bacterial aggregation, encountered in the direct separation of bacteria. The separation of the flavonoid kaempferide was realized by capillary zone electrophoresis, and kaempferide in Kaempferia galanga was determined. Kaempferide and the extract of Kaempferia galanga were applied in the antibacterial tests separately. Using the developed method, the bacterial growth curves and the curves of the kaempferide concentration were studied respectively. The results showed that both kaempferide and Kaempferia galanga could effectively inhibit the bacteria. The inhibitory effect on the terrestrial bacteria Escherichia coli, and Staphylococcus aureus was better than that on the marine bacterium Vibrio parahaemolyticus.

While traditional dielectrophoretic methods for nanoparticle enrichment and filtration are versatile and selective, they struggle to handle higher throughput applications. To address this challenge and enhance the practical application of dielectrophoresis, we propose an innovative design for porous sandwiched nanofiber electrodes. The electrode is fabricated through a simple process involving the electrospinning of nanofibers with a diameter of 216 ± 28 nm and mat thickness of around 70 µm, followed by the deposition of a thin chromium/gold layer (approximately 140 nm thick) on both sides. This process ensures no electrical short circuit occurs between the electrodes, and it maintains a sheet resistance of 7.19 Ω/□. The resulting significant electric field gradients are capable of trapping nanoparticles with diameters of 100 nm and 40 nm. The structure's sub-micrometer features and large active surface area allow for trapping of nanoparticles at a flow rate of 3.6 mL/h. To evaluate the effects of applied voltage and volumetric flow rate, we conducted experiments with constant voltage while varying the flow rate and constant flow rate while varying the voltage. Our findings indicate that trapping performance improves with higher AC voltage but decreases at higher flow rates. These insights are crucial for optimizing parameters for large-scale nanoparticle enrichment and filtration. This proof-of-concept study for flow through dielectrophoresis of nanoparticles paves the way for a device suitable for large-scale sample processing and higher throughput/separation efficiency in practical settings.

Tumor–macrophage interactions play a key role in various physiological and pathological processes, such as angiogenesis, immune suppression, and extracellular matrix remodeling. In this study, a biomimetic microfluidic chip was developed to simulate the immune microenvironment of glioma through the co-culture of glioma cells and macrophages in a three-dimensional (3D) matrix. Glioma cells were embedded in collagen I solution after forming spheroids in the microwell array chip and subsequently co-cultured with macrophages in different channels. This chip enabled the real-time monitoring of morphological changes in macrophages, the invasion of glioma cell spheroids, and molecular interactions between different cell types. Two distinct cell types could be extracted and isolated in situ for subsequent molecular biological detection, such as Western blotting or qPCR. The results demonstrated that glioma cell spheroids significantly enhanced invasiveness in the presence of macrophages. Moreover, the phenotype of macrophages altered from M0 to M2 (tumor-supportive) under the influence of tumor cells. The molecular mechanism mediating this reciprocal process was extensively explored. It is believed that this 3D microfluidic tumor model could serve as a useful tool for studying the biological properties of the glioma microenvironment. In addition, a more comprehensive understanding of the mechanisms involved in glioma metastasis could be obtained, especially of how tumor inflammatory cells, including tumor-associated macrophages (TAM), affect invasion process.