ELECTROPHORESIS最新文献

Fully characterizing subtle differences in biologically important particles—including peptides, proteins, protein complexes, exosomes, viruses, organelles, and cells—is essential, as any alteration can impact their function. Detailed bioparticle characterization has broad implications for biomedical engineering, health care, food science, astrobiology, environmental studies, and microbiology. Dielectrophoresis (DEP) generates distinct forces based on subtle structural differences between bioparticles and has the potential to enable full characterization by quantifying the DEP response of a particle. However, current DEP techniques primarily rely on particle trapping, which presents limitations, particularly for nanoparticles. In contrast, streaming-based DEP measurement techniques remain largely unexplored. Here, we introduce a streaming-based microfluidic method inspired by the (inverse) classical scattering problem in physics. Using a custom insulator-based DEP microchannel (iDEP), the DEP susceptibility of a particle is quantified based on its predictable deflection magnitude. We demonstrate the feasibility of this approach for negative DEP using finite element analysis to conduct numerical scattering experiments on representative nanoparticles. To fully capture diffusion effects, we solved the steady-state Smoluchowski advection–diffusion equation to obtain concentration fields in the microchannel and extract realistic scattering profiles. Additionally, deterministic particle trajectories, computed in the absence of diffusion, were analyzed using streamline analysis to support the advection–diffusion results. Our results indicate that, under optimal conditions, the prototype iDEP microchannel approaches the necessary sensitivity for protein DEP characterization, even when diffusion is included. Like existing iDEP devices, a real iDEP scattering instrument is expected to be easy and inexpensive to operate. Combined with the straightforward processing and interpretation of the scattering data, the iDEP scattering technique has the potential to enable high-throughput, accurate bioparticle characterization.

Solid-state nanopores (SSNPs) are progressively gaining importance in biomolecular sensing and ionic circuit applications. Unlocking their full potential, however, requires the development of fabrication techniques that enable precise control over their sizes and shapes. Electron-beam (EB) shrinking provides precise, real-time feedback and is ideally suited to address these requirements. However, it necessitates an initial pore diameter smaller than the membrane thickness for effective shrinking without material addition. Typical focused ion beam (FIB)-drilled pores in silicon nitride membranes often fail to meet these requirements. Alternative efforts towards mitigating these bottlenecks through deploying hydrocarbon-mediated EB shrinkage face challenges due to uncontrolled carbon contamination or a lack thereof in cleaner transmission electron microscope (TEM) chambers. To address these challenges, here we report an alternative approach of high-precision hydrocarbon-mediated EB shrinking with hydrocarbons sourced externally through controlled surface reactions on exposure to ethanol. This provides several decisive advantages, including the reduction of pore diameters much larger than the membrane thickness and controlled shrinking in cleaner environments without contaminations. These measures accelerate nanopore fabrication, improve its predictability by eliminating the dependence on variable carbon contamination in vacuum chambers, and provide high-resolution live feedback during dimension tuning. As a result, our method supports the large-scale production of nanopores with analyte-specific, tuneable dimensions. This capability is particularly imperative for low-noise biomolecular sequencing applications that leverage electrically-modulated transport and sensing over nanoscales. These features could pave the way for the broader application of SSNPs, addressing long-standing challenges in their fabrication and functionalisation that remained unresolved thus far.

The MC1R gene, which is responsible for most cases of red hair, affects other hair and skin colours and contributes to differences in pain sensitivity and consists of a single exon with a very high level of allelic heterogeneity. In this research, we show that the Oxford Nanopore Technology (ONT) offers a good alternative to study the MC1R sequence variation. MinION was used to sequence the 1590 bp MC1R exon and minimal promoter in a cohort of 126 subjects, including 65 red-haired individuals, using the FLO-MIN106 (R9.4) chemistry. Assigned DNA variants were validated using Ion Torrent technology provided with Ion Xpress Plus Fragment Library Kit and the Personal Genome Machine^TM (PGM^TM). We show that the use of the latest sequencing kit V14 together with the FLO-MIN114 (R10.4.1) flow cell has eliminated the systematic errors observed with the previous chemistry and allowed reliable detection of short indels important for phenotypic inference. Importantly, the use of the algorithm implemented in the EPI2ME software enabled convenient and accurate read-based phase determination which can be useful in data interpretation.

Various forms of dielectrophoresis and higher order electrokinetic effects are being increasingly investigated and used to precisely and accurately manipulate micro and nanoparticles within microfluidic devices. The types of particles span ∼10 nm to hundreds of microns in diameter and are composed of minerals, polymers, biological materials, and complex mixtures. Some studies focused on the selective isolation and concentration of purified particles countering negative dielectrophoretic forces against flow and electrophoretic effects. Similar studies are presented here examining the behaviors of small inorganic particles (10 nm diameter) where their collective actions are inconsistent with negative dielectrophoretic effects and were consistent overall with positive dielectrophoresis (DEP). Positive DEP can account for some of the observed phenomena, particularly the deflection of large particle aggregates, which are rapidly accelerated through microchannel constrictions and then pulled back toward the constrictions against the direction of electroosmotic flow. Nevertheless, the dynamic complexity of the observed nanoparticle structures suggests that a myriad of electrostatic and possibly hydrodynamic forces, including both particle–particle and particle–device interactions, may be involved.