ELECTROPHORESIS最新文献

Monoclonal antibodies (mAbs) are complex therapeutic proteins exhibiting heterogeneity due to post-translational modifications, making charge variant analysis essential for defining critical quality attributes. Although ion-exchange chromatography and capillary isoelectric focusing are established techniques, they require extensive optimisation, whereas the widely adopted ε-aminocaproic acid-based capillary zone electrophoresis (CZE) (eACA-CZE) method provides a simpler, robust platform. However, its performance can be limited for mAbs whose charge profiles or isoelectric point (pI) values fall outside the method's optimal range. To expand CZE capabilities while maintaining a platform approach, we developed a series of background electrolytes (BGEs) spanning a range of pH values, polyamine concentrations and buffering capacities. These BGEs were formulated for chemical compatibility, long-term stability and fixed-component composition to ensure consistent pH and reproducible currents. Using a pH 5.7 BGE as the starting point, key components were optimised across the full buffer set. A multivariate approach showed that combining these buffers enhanced resolution, resolving additional impurity peaks in 9 of 10 mAbs. This work establishes a flexible toolkit for screening and optimising charge-variant resolution across diverse mAbs.

The use of dielectrophoresis (DEP) for the manipulation of electrically polarizable particles has received a lot of interest in the past decades. Theories relying on the Clausius-Mossotti (CM) factor describe the DEP behavior of macroscale particles with great accuracy. However, under nanoscale conditions, these classical CM factor theories can break down. Therefore, experimental characterization of particle polarizabilities is of utmost importance. We present an integrated experimental-computational methodology that allows the quantification of effective particle polarizabilities from DEP experiments. The methodology is based on the comparison of experimental and simulated concentration profiles of fluorescent nanoparticles captured by a set of electrodes. We obtain effective particle polarizabilities for 52 and 105 nm particles that agree well with values predicted by CM theory. The considered particles hence serve as a calibration model for our approach, demonstrating that realistic particle polarizabilities can be obtained and paving the way for polarizability quantification for particles where existing theories are inadequate.

In complex systems, a universal and effective method for accurately determining the dissociation constant (K_d) of drug-protein interactions (DPI) has not been identified yet. In this study, a novel biomimetic affinity capillary electrochromatography (ACEC) platform was developed. Polydopamine (PDA) was employed not only as a coating material but also as an immobilization agent for human serum albumin (HSA), resulting in the fabrication of the PDA/PDA/HSA@capillary. A series of characterization experiments on the PDA/PDA/HSA@capillary showed that PDA was successfully coated on the inner wall of the capillary, and HSA was also successfully immobilized on the capillary column. The optimal concentration of PDA for preparing the PDA/PDA/HSA@capillary was determined to be 0.8 mg mL^-1, and the optimal concentration for immobilizing HSA was 0.25 mM, respectively. Moreover, the PDA/PDA/HSA@capillary showed good separation effects in the complex systems of different drugs. Furthermore, the electrophoretic performance of individual and mixed samples was compared across different capillary columns. Through interaction studies between proteins and compounds, the K_d value of rutin was 1.09 × 10³ mol L^-1 that of quercitrin was 2.78 × 10³ mol L^-1, and that of quercetin was 7.44 × 10⁴ mol L^{- 1}, aligning with results from other established methods. The method was applied to the Sophora japonica extract, and the K_d was consistent with the rutin in the mixed system. Reproducibility studies demonstrated that high separation efficiency was maintained even after 50 consecutive runs. By integrating biomimetic material science with chromatographic innovation, this work overcomes critical bottlenecks in traditional affinity capillary electrophoresis (ACE), providing a universal tool for high-throughput drug screening and structure-guided therapeutic design.

We present a novel paper-based nucleic acid amplification (NAA) technique using electrokinetic nucleic acid amplification (E-NAAMP). In E-NAAMP, a high radio frequency (RF) potential is applied across a conductive aqueous sample to induce an Ohmic current and drive the sample temperature to increase by Joule heating. Using this RF approach, we investigate the ability to induce E-NAAMP in pressurized paper-based microfluidic channels. We use the microfluidic pressure-in-paper (µPiP) method to encapsulate synthetic and natural fiber-based paper channels between thin sheets of polydimethylsiloxane (PDMS) with two strips of conductive PDMS that have been infused with carbon black (PDMS-CB) to act as electrodes in contact with the paper channels. A high-frequency (38 MHz) voltage is applied across a conductive NAA sample via the PDMS-CB electrodes to generate Joule heating within the paper structure. Here, we show that µPiP-based E-NAAMP can amplify NAs using the loop-mediated isothermal amplification (LAMP) reaction. We first investigate the pore-scale temperature profile numerically by solving the relevant energy transport equations within digitized paper fiber domains obtained using micro-computed tomography (micro-CT) scans. We compare these temperature-voltage predictions to those measured experimentally and demonstrate good agreement, suggesting that RF Joule heating is a viable method for electrokinetically heating paper-based microfluidic platforms. We next examine the effect of a porous substrate on NAA and demonstrate that the carrier protein, bovine serum albumin (BSA), is required for paper-based NAA reactions by preventing polymerase adsorption within the paper structure. Finally, we show successful NAA in paper using E-NAAMP with multiple paper fiber types while also further demonstrating BSA's necessity for paper E-NAAMP success. Our results demonstrate that paper-based microfluidic NAA using Joule heating is a viable alternative to traditional microfluidic NAA devices by offering substantial heating element miniaturization and decreased fabrication complexity when compared to both traditional and Joule-heated microfluidic NAA devices.

Phenolic preservatives are critical excipients in biopharmaceutical formulations such as insulin and its analogs. The determination of these analytes in insulin formulations and human plasma is crucial for various applications. This study reports the first use of a method combining salting-out assisted liquid-liquid extraction (SALLE) with capillary zone electrophoresis (CZE). The CZE-based separation of phenolic preservatives remains particularly challenging, as the alkaline conditions required for adequate resolution lead to elevated electric current and accelerated capillary deterioration. Analytical parameters, including the nature of the background electrolyte (BGE) and ionic strength, were optimized to ensure reliable separation of phenol, m-cresol, and the internal standard. SALLE was performed using acetonitrile (ACN) as the extraction solvent and ammonium acetate as the salting-out agent. CZE separation was completed in less than 6 min using a carbonate buffer at pH 10.3. The SALLE procedure yielded good recoveries (96%-102%) and repeatability (relative standard deviation [RSD] for concentration determination < 5.5%). The SALLE-CZE-UV methodology showed excellent linearity (r² > 0.994) and repeatability (RSD for corrected migration times: 0.4% for phenol and 0.5% for m-cresol; RSD for the ratio of corrected peak areas [analyte/internal standard]: 2.6% for phenol and 1.8% for m-cresol). Moreover, appropriate limits of detection (LOD) and quantification (LOQ) were obtained for insulin formulations in line with regulatory requirements. In addition, suitable LOD and LOQ (0.014; 0.047 g.L^-1 for phenol and 0.007; 0.022 g.L^-1 for m-cresol) were obtained for spiked human plasma.

Capillary electrophoresis (CE) often provides superior separation of macromolecules such as monoclonal antibodies (mAbs), a major biopharmaceutical class, compared with liquid chromatography. However, electropherograms frequently exhibit complex baselines and peak shapes that are not reliably handled by integration algorithms designed for chromatographic data, and manual integration is often required. Many concepts have been proposed to improve peak integration, ranging from incremental algorithmic refinements and signal-to-noise (S/N)-based approaches to artificial intelligence (AI)-driven strategies, but objective performance comparisons are not possible without shared reference data sets and agreed peak limits. To address this gap, we initiated a multinational collaboration involving industrial and academic laboratories to create a comprehensive reference data set for CE peak integration. A total of 227 challenging and practically relevant electropherograms were collected from diverse applications, converted to a standardized format, and independently integrated by multiple experts. Using dedicated software tools and a structured consensus process, mutually accepted reference integration limits were established for each data set. These reference electropherograms, together with the underlying integration rules, are now made available to the scientific community. Analysis of the reference data set identified general principles for reliable peak integration, including the importance of standardized zoom levels and consistent handling of small peaks near the noise level. The data set provides a common foundation for benchmarking commercial chromatography data systems (CDS) and for developing and validating new algorithmic and AI-based integration methods. We expect this work to speed up the development of practical, automated integration strategies for CE and that these core concepts can be applied to other separation techniques.

This study presents a capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF) method for analyzing topoisomers of large plasmid DNA (up to 19 kbp) using an uncoated capillary. Separation of large plasmid isoforms, particularly the open circular (OC) form, poses significant challenges in gel buffer formulation. To address this, we systematically investigated the effects of multiple parameters-including polymer type, fluorescent dye selection, stir bar geometry, stirring speed and duration, gel volume, container dimensions, and technique for polymer addition-on separation performance. The optimized gel buffer composition was 0.9% polyethylene oxide (PEO), 0.02% SYBR Green, 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 1 mM EDTA, and 20 mM NaCl at pH 6.25. Replacing hydroxypropyl methyl cellulose (HPMC) gel with PEO gel significantly improved OC isoform separation. Furthermore, substituting SYBR Gold with SYBR Green enhanced the robustness of OC separation and yielded a 4- to 8-fold increase in sensitivity. The PEO gel preparation process was successfully scaled and optimized at the 100 mL level. With consistent capillary cleaning and storage protocols, the method demonstrated excellent reproducibility over 150 consecutive runs.

Generating monodisperse droplets with tunable concentrations is essential for a wide range of microfluidic applications. We present a platform that overcomes the trade-off between concentration range and droplet size stability using a passive, water-head-driven system with asymmetric fluidic resistances. This design effectively decouples concentration tuning from droplet generation within the tested regime by stabilizing the junction pressure to ensure a steady total aqueous flow rate. We demonstrate theoretically and experimentally that this results in monodisperse droplets of nearly constant size across the programmed concentration range. Concurrently, by adjusting a single passive pressure inlet, the system achieves continuous control of the concentration ratio over a five-order-of-magnitude dynamic range. This robust, simple method eliminates complex pumps, offering a powerful tool for high-throughput screening and combinatorial studies.

mRNA-based vaccines and self-amplifying mRNA (saRNA) have gained growing attention for disease prevention and treatment, but precise detection of low-concentration RNA (including mRNA-lipid nanoparticles, mRNA-LNPs) stability and integrity remains challenging-limiting quality control of RNA-based therapeutics. Capillary electrophoresis (CE)-based instruments show potential, yet suitable concentration strategies for low-abundance, labile RNAs are lacking. This study established two distinct concentration methods (freeze-drying and ultrafiltration) applicable to mRNA, mRNA-LNPs, and saRNA formulations, enabling the conversion of dilute solutions to high-concentration preparations while preserving the structural and molecular integrity of the target nucleic acids. Subsequent stability evaluations and conventional high-performance liquid chromatography analyses of the concentrated products verified their superior storage stability and strong practical applicability. This article aims to reduce the difficulty of RNA integrity assessment and improve the accuracy of detecting various low-concentration RNA samples that may arise in the future.

This study describes the use of microchip micellar electrokinetic chromatography (MEKC) integrated with capacitively coupled contactless conductivity detection (C⁴D) for the determination of monoethylene glycol (MEG) in gas condensate samples. The samples were subjected to a liquid-liquid extraction step and then analyzed by chip-based MEKC-C⁴D. For this purpose, sodium dodecyl sulfate (SDS) was used as a surfactant at a concentration of 30 mmol L^-1 added in 50 mmol L^-1 phosphate (pH = 9.0). Samples were introduced into microchannels through floating injection mode by applying a voltage of 600 V during 10 s. Separations were performed under an electric field of 82 V cm^-1 and monitored by C⁴D measurements recorded applying a 1200-kHz frequency sinusoidal wave with 20-V_pp excitation voltage. The proposed methodology employing MEKC-C⁴D revealed a linear behavior (r² ≥ 0.99) in the MEG concentration range between 150-450 µmol L^-1 and LOD equal to 33 µmol L^-1. Three gas condensate samples were then analyzed, and the achieved MEG concentration values ranged from 173 to 213 µmol L^-1. Recovery experiments provided values between 89 and 102%. Based on the results reported in this study, MEKC-C⁴D devices have demonstrated to be a promising and ecological analytical tool for MEG analysis with huge potential for in-field applications.