Single-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides the closest view of the status quo in the cell, studying the metabolome at single-cell resolution may unravel said heterogeneity. A challenge in single-cell metabolome analysis is that metabolites cannot be amplified, so one needs to deal with picolitre volumes and a wide range of analyte concentrations. Due to high sensitivity and resolution, MS is preferred in single-cell metabolomics. Large numbers of cells need to be analysed for proper statistics; this requires high-throughput analysis, and hence automation of the analytical workflow. Significant advances in (micro)sampling methods, CE and ion mobility spectrometry have been made, some of which have been applied in high-throughput analyses. Microfluidics has enabled an automation of cell picking and metabolite extraction; image recognition has enabled automated cell identification. Many techniques have been used for data analysis, varying from conventional techniques to novel combinations of advanced chemometric approaches. Steps have been set in making data more findable, accessible, interoperable and reusable, but significant opportunities for improvement remain. Herein, advances in single-cell analysis workflows and data analysis are discussed, and recommendations are made based on the experimental goal.

This review article brings a comprehensive survey of developments and applications of high-performance capillary and microchip electromigration methods (zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical characterization of peptides in the period from 2021 up to ca. the middle of 2023. Progress in the study of electromigration properties of peptides and various aspects of their analysis, such as sample preparation, adsorption suppression, electroosmotic flow regulation, and detection, are presented. New developments in the particular capillary electromigration methods are demonstrated, and several types of their applications are reported. They cover qualitative and quantitative analysis of synthetic or isolated peptides and determination of peptides in complex biomatrices, peptide profiling of biofluids and tissues, and monitoring of chemical and enzymatic reactions and physicochemical changes of peptides. They include also amino acid and sequence analysis of peptides, peptide mapping of proteins, separation of stereoisomers of peptides, and their chiral analyses. In addition, micropreparative separations and physicochemical characterization of peptides and their interactions with other (bio)molecules by the above CE methods are described.

Enantioseparation by the electromigration-based method is well-established and widely discussed in the literature. Electrophoretic strategies have been used to baseline resolve complex enantiomeric mixtures, typically using a selector substance into the background electrolyte (BGE) from capillaries to microchips. Along with developing new materials/substances for enantioseparations, it is the concern about the green analytical chemistry (GAC) principles for method development and application. This review article brings a last decade's update on the publications involving enantioseparation by electrophoresis for capillary and microchip systems. It also brings a critical discussion on GAC principles and new green metrics in the context of developing an enantioseparation method. Chemical and green features of native and modified cyclodextrins are discussed. Still, given the employment of greener substances, ionic liquids and deep-eutectic solvents are highlighted, and some new selectors are proposed. For all the mentioned selectors, green features about their production, application, and disposal are considered. Sample preparation and BGE composition in GAC perspective, as well as greener derivatization possibilities, were also addressed. Therefore, one of the goals of this review is to aid the electrophoretic researchers to look where they have not.

High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min-1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC-LIN and LIN-SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.

Proteins are important molecules involved in an immensely large number of biological processes. Being capable of manipulating proteins is critical for developing reliable and affordable techniques to analyze and/or detect them. Such techniques would enable the production of therapeutic agents for the treatment of diseases or other biotechnological applications (e.g., bioreactors or biocatalysis). Microfluidic technology represents a potential solution to protein manipulation challenges because of the diverse phenomena that can be exploited to achieve micro- and nanoparticle manipulation. In this review, we discuss recent contributions made in the field of protein manipulation in microfluidic systems using different physicochemical principles and techniques, some of which are miniaturized versions of already established macro-scale techniques.

We propose a novel technique, consistent with the induced charge electrokinetic (ICEK) phenomenon, for the efficient mixing of solute species at a microfluidic scale. A nonuniform bipolar electric double layer develops in the presence of an external electric field over a polarizable object is better known as the ICEK phenomenon. This ICEK is one of the most favorable techniques preferred for enhanced solute mixing in on-chip microfluidic platforms. In the purview of the ICEK phenomenon, instead of using perfectly conducting polarizable objects, for the first time in this study, we employ polarizable dielectric objects of different sizes and shapes for efficient mixing of solute species. We show that different types of vortices developed in the flow pathway adjacent to the polarizable dielectric blocks help in yielding efficient mixing in the proposed configuration. The novelty of our work is embellished in two different perspectives, that is, first, concentrating on the influences of the physical properties of the polarizable dielectric block on the underlying mixing, and, second, focusing on their sizes, shapes, and the arrangements in tuning the underlying mixing phenomena.

Milk is a rich source of biologically important proteins and peptides. In addition, milk contains a variety of extracellular vesicles (EVs), including exosomes, that carry their own proteome cargo. EVs are essential for cell-cell communication and modulation of biological processes. They act as nature carriers of bioactive proteins/peptides in targeted delivery during various physiological and pathological conditions. Identification of the proteins and protein-derived peptides in milk and EVs and recognition of their biological activities and functions had a tremendous impact on food industry, medicine research, and clinical applications. Advanced separation methods, mass spectrometry (MS)-based proteomic approaches and innovative biostatistical procedures allowed for characterization of milk protein isoforms, genetic/splice variants, posttranslational modifications and their key roles, and contributed to novel discoveries. This review article discusses recently published developments in separation and identification of bioactive proteins/peptides from milk and milk EVs, including MS-based proteomic approaches.

Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16 kbp in size.

Recovery of noble materials from waste is essential for industries around the globe. Dielectrophoretic (DEP) filtration, an electrically switchable particle separation technique, can be applied to tackle this challenge. It is highly selective regarding particle size, material or shape. Expanding the scope of DEP towards high throughput and improving the trapping efficiency are vital to make DEP a viable robust alternative to conventional separation methods. DEP filtration works by selective immobilisation of particles in a porous medium by the action of an inhomogeneous electric field. The field inhomogeneity comes from scattering an electric field at the phase boundary between the particle suspension and the filter surface. In this article, we show how the filter structure affects the DEP separation. We study fixed bed filters of three different grain types and find that the morphology of the grains highly influences the DEP filter efficiency. Specifically, grains with irregular surface structure and high perceived angularity show high separation efficiency. We believe these insights into the design of DEP filtration will pave the way towards its application in, for example, the recovery of valuable materials from electronic waste dust.

Thermophoresis of spheroidal colloids in aqueous media under the thermal conductivity effect is analyzed. The thermophoretic velocity and the thermodiffusion coefficient of spheroidal colloids have been formulated for extremely thin electric double layer (EDL) cases. Furthermore, a numerical thermophoretic model is built for arbitrary EDL thickness cases. The parametric studies show that the thermal conductivity mismatch of particle and liquid gives rise to a nonlinear temperature region around the spheroid, with the thickness close to the minor semiaxis. When the EDL region is thin relative to such nonlinear temperature region, the thermal conductivity effect on the thermophoresis of spheroidal colloids is significant, which strongly depends on the ratio of the minor semiaxis to the EDL thickness, the thermal conductivity ratio of particle to liquid, and the particle aspect ratio. Finally, to estimate the thermodiffusion coefficient of spheroidal colloids with arbitrary thermal conductivity, electrolyte concentration, and particle shape, the average dimensionless axial temperature gradient on the spheroidal equator plane in the EDL region is proposed.

Active magnetic regulation is an emerging subject due to the special and programmable wettability of the sessile ferrofluid droplet. The interaction between liquid and externally applied magnetic field gives rise to controllable spreading and thus evaporation. This work reports the experimental and numerical results of the natural evaporation of a ferrofluid droplet under the effect of a nonuniform magnetic field. The evaporation process of droplets is described into two stages in terms of the geometric distortion and the appearance of the deposition pattern. The presence of the magnetic field leads to a transition of droplet drying from the disk shape with a ring to multiple peaks. A numerical model is established to simulate the evaporation process of ferrofluid droplets with the arbitrary Lagrangian-Eulerian method for tracking droplet deformation. The increasing magnetic flux could effectively enlarge the contact radius and enhance the internal flow of the ferrofluid droplet, thus promoting the evaporation process. The numerical results are verified by comparing the droplet geometry deformation with the experimental results. In both the numerical and experimental investigations, the externally applied magnetic field shortens the process of ferrofluid droplet evaporation. The design and optimization of the magnetic field play an important role in regulating ferrofluid droplet evaporation, which in turn facilitates technological advances in industries such as evaporative cooling and inkjet printing.

In recent years, researchers have made significant strides in understanding the ion transport characteristics of nanochannels, resulting in the development of various materials, modifications, and shapes of nano ion channel membranes. The aim is to create a nanochannel membrane with optimal ion transport properties and high stability by adjusting factors, such as channel size, surface charge, and wettability. However, during the nanochannel film fabrication process, controlling the geometric structures of nanochannels can be challenging. Therefore, exploring the stability of nanochannel performance under different geometric structures has become an essential aspect of nanochannel design. This article focuses on the study of cylindrical nanochannel structures, which are categorized based on the different methods for generating bipolar surface charges on the channel's inner surface, either through pH gradient effects or different material types. Through these two approaches, the study designed and analyzed the stability of ion transport characteristics in two nanochannel models under varying geometric structures. Our findings indicate that nanochannels with bipolar properties generated through pH gradients demonstrate more stable ion selection, whereas nanochannels with bipolar properties generated through different materials show stronger stability in ion rectification. This conclusion provides a theoretical foundation for future nanochannel designs.

In order to localize heat production and drug activation, it is possible for drug delivery to make use of nanofluids containing thermal radiation. By limiting the amount of medication that is administered to healthy tissues, this approach increases drug distribution. We explore the effect that thermal radiation has on the flow of a ternary-hybrid nanofluid composed of titanium oxide (TiO2 ), silica (SiO2 ), and aluminum oxide (AI2 O3 ). The base liquid that we use for our Carreau constitutive model is blood. Entropy and electroosmosis are both taken into account when the conduit is connected to the battery terminals outside. Following the step of translating the observation model into a wave frame, the physical restrictions of the lubrication theory are used in order to provide a more complete explanation for the wave occurrences. In this work, shooting is used to simulate boundary value issues that are solved with Mathematica NDSolve. The production of the least amount of entropy and a rise in thermodynamic efficiency are achieved by the motion of cilia and elastic electroosmotic pumping. It is also observed that heat transfer is proportional to the length of cilia. Nusselt number is increased by large cilia but skin friction got a reduction.

The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA, BIA Separations). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1 mL CIMmultus QA columns to the 8 L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.

Separation and identification of chiral molecules is a topic widely discussed in the literature and of fundamental importance, especially in the pharmaceutical and food fields, both from industrial and laboratory points of view. Several techniques are used to carry out these analyses, but high-performance liquid chromatography is often the "gold standard." The high costs of chiral columns, necessary for this technique, led researchers to look for an alternative, and capillary electrophoresis (CE) is a technique capable of overcoming some of the disadvantages of liquid chromatography, often providing comparable results in terms of sensitivity and robustness. We addressed this topic, already widely discussed in the literature, providing an overview of the last 6 years of the most frequent and recent applications of CE. To make the manuscript more effective, we decided to divide it into paragraphs that represent the main field of application, from enantioseparation in complex matrices (pharmacokinetic studies or toxicological dosage of drugs, analysis of environmental pollutants, and analyses of foods) to quality control analyses on pharmaceutical formulas. About these, which are the fields of most meaningful use, we mentioned some of the most innovative and performing methods, with a look to the future on the application of new materials used, such as chiral selectors, that can make these types of analyses accessible to all, reducing cost, time, and excessive use of toxic solvents.

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15-30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing-based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2-4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.

Electrophoresis of a dielectric fluid droplet with constant surface charge density is investigated theoretically in this study. A pseudo-spectral method based on Chebyshev polynomials is adopted to solve the governing electrokinetic equations. It is found, among other things, that the larger the electrolyte strength in the ambient solution is, the slower the droplet moves in general. This is due to the strong screening effect of the large amount of indifferent counterions in the neighborhood of the droplet, with no reinforcement of potential-determining ions adsorbing to the droplet surface. The droplet comes to a complete halt eventually. Critical points are discovered for highly charged droplets, at which the droplet surface becomes immobile and the interior fluid stops recirculating. The droplet moves like a rigid particle with constant mobility regardless of its viscosity, a situation referred to as the "solidification phenomenon." The deadlock between the spinning motions on the charged droplet surface induced by the electric driving force and the hydrodynamic driving force respectively is responsible for this peculiar phenomenon. This is also observed for a dielectric droplet with constant surface electric potential. We demonstrate here that it occurs in the constant surface charge density situation as well.

Much progress has been made in the electrokinetic phenomena inside nanochannels in the last decades. As the dimensions of the nanochannels are compatible to that of the electric double layer (EDL), the electrokinetics inside nanochannels indicate many unexpected behaviors, which show great potential in the fields of material science, biology, and chemistry. This review summarizes the recent development of nanofluidic electrokinetics in both fundamental and applied research. First, the techniques for constructing nanochannels are introduced to give a guideline for choosing the optimal fabrication technique based on the specific feature of the nanochannel. Then, the theories and experimental investigations of the EDL, electroosmotic flow, and electrophoresis of nanoparticles inside the nanochannels are discussed. Furthermore, the applications of nanofluidic electrokinetics in iontronics, sensing, and biomolecule separation fields are summarized. In Section 5, some critical challenges and the perspective on the future development of nanofluidic electrokinetics are briefly proposed.

At present, there is still limited report on the electrokinetic (EK) behavior of bioparticles at the interface of an aqueous two-phase system. In this paper, the EK motion and viability assessment of live algae mixed with the NaClO treated dead algae were carried out at the interface formed by polyethylene glycol (PEG)-rich phase and dextran (DEX)-rich phase in a straight microchannel. The experimental results show that both the live and dead algae at the PEG-DEX interface migrate from the negative electrode to the positive electrode, and the EK velocity of live algae at the interface is slightly larger than that of the dead ones with similar diameters. For either live or dead algae, the EK velocity at the interface decreases with the increase in diameter. A size-velocity curve was used to evaluate the viability of the algae. As most of the microorganisms in ballast water are algae, the method in this paper provides a promising way to detect and evaluate the live microorganism in treated ballast water of a ship.