Gradient Separation Research Articles

Tuning Nanochannel Microenvironments of a Thermoresponsive MXene Membrane for Mixed Molecule Gradient Separation.

Drawing inspiration from dynamic biological ion channels, researchers have developed various artificial membranes featuring responsive nanochannels. Typically, these membranes modify mass transport behaviors by manipulating the responsive layer on the inner surfaces of the intrinsic layer. In this study, we build two-dimensional lamellar membranes composed of titanium carbide MXene and poly(N-isopropylacrylamide), endowed with dual-level regulatable nanochannels, achieved through adjustments of nanochannel microenvironments. The size of these two-dimensional nanochannels can be altered by both the thermoresponsive polymer layer and the intrinsic MXene layer that could undergo spontaneous oxidation. The multilevel regulation strategy substantially enhances the molecular selectivity of MXene separation membranes, which is further applied for precise gradient separation toward multiple molecules. This advancement showcases the versatility and transformative capabilities of responsive nanochannel technology, setting the stage for innovative developments in diverse fields.

Exploring the utility of complementary separations in liquid chromatography

An alternative strategy is explored for the separation of samples by liquid chromatography (LC). Unlike traditional approaches that aim to resolve all components in a given sample within a single LC separation, the proposed strategy uses two or more distinct separations carried out with a different gradient program and/or using different separation chemistries i.e., a different set of mobile and stationary phase. This set of complementary incomplete separations (CIS) is selected such that each component is at least fully resolved once, meaning the most critical pairs of each individual separation can be left unseparated. This allows for a significant time saving per separation. To investigate whether such an approach can lead to overall shorter analysis times than is possible with the fastest single-run gradient separation, a comprehensive in-silico study covering a statistically significant number of samples is undertaken. The investigation shows that, for the presently considered sample sets and chemistries, CIS has a substantially higher probability, about two times greater for the simplest samples considered in this work and as much as 30 times greater for more complex samples, to fully resolve an unknown sample compared to a single gradient separation. Comparing separation speeds, the CIS approach can achieve complete sample resolution on average approximately four times faster than a single separation. Our findings thus demonstrate the potential of CIS in enhancing separation efficiency and offer insights regarding their use for solving analytical challenges.

Impregnation, dehydration and crosslinking of responsive organoboron polymer to generate smart gating membrane for multimolecular gradient separation

Responsive materials have garnered increasing attention in the membrane separation field. However, fabricating smart gating membranes with tunable pore sizes to separate complex systems remains challenging. Herein, gating membranes with boroxine skeleton and temperature-tunable pores were successfully fabricated by fixing lower critical solution temperature (LCST)-type organoboron polymers (poly(N-isopropylacrylamide-co-glycidyl methacrylate/3-aminophenylboronic acid), PNG-APBA) onto the membrane surface via "impregnation-dehydration-crosslinking" strategy. The conformational behavior of the NIPAM-containing polymer chains (shrinking above LCST-stretching below LCST) serves as a functional gate, enabling the membranes to achieve reversibly tunable pore sizes and surface properties. The modified membranes exhibit gradient separation capabilities for small/medium/large molecules in complex polymer systems through temperature-tunable channels. The tunable pores also provided a potential tool for the high-selectivity separation of mixed proteins, such as lysozyme (LZM) and hemoglobin (Hb). Notably, the conformational behavior of the polymer chains endowed the membranes with excellent self-cleaning ability (FRR> 99.5%), while the boroxine network enhanced the grafting stability of the polymer chains, ensuring effective reversibility and repeatability of membranes.

Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle

Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of Neospora caninum obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of N. caninum, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.

Precise gradient separation of nanoparticles using reduced graphene oxide-polyoxomolybdate membranes

Precise gradient separation of nanoparticles using reduced graphene oxide-polyoxomolybdate membranes

Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical samples.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline

BackgroundKlebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear.MethodsTo isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis.ResultsK. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group.ConclusionsThis study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Gradient Separation and Recovery of Pb, Se, Cu, and Hg from Acid Sludge by a Sustainable Hydrometallurgical Process

Gradient Separation and Recovery of Pb, Se, Cu, and Hg from Acid Sludge by a Sustainable Hydrometallurgical Process

Streamlined integrated protein isoelectric focusing using microfluidic paper-based device

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Structural Phase Separation of Membranes and Fibers.

Lipid membranes interact with protein filaments on a superstructural level such that they may colocalize or spatially segregate in a living cell, whereas higher-order organization of membranes and fibers is less well explored in artificial systems. Herein, we report on the structural separation of a dispersed, membranous phase and a continuous, fibrous phase in a synthetic system. Systematic characterization of its thermodynamics and kinetics uncovers a physical principle governing phase separation: Interlamellar repulsion, favoring expansion of the membranous phase, is balanced by fibrous network elasticity, preferring the opposite. A direct consequence of this principle is the spatial addressability of the phase separation, preferably localized to soft regions of the fibrous network. Guided by this principle, we design a fibrous network with different spatial heterogeneity to modulate the phase separation, realizing a "memory" effect, patterned separation, and gradient separation. The current spatially addressable phase separation is in great contrast to the conventional ones, in which nucleation is difficult to predict or control. The fact that the membranous and fibrous phases compete for space has implications for the intracellular interactions between endoplasmic reticulum membranes and cytoskeletal filaments.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Extracellular and intracellular inflammatory response of immune cells to radiation exposure

Background: Exposure to low doses of ionizing radiation is associated with increased risk of cardiovascular disease (CVD) in a dose-dependent manner. A person exposed to low doses of radiation have a 27-29% risk of developing and dying from CVD. Low doses of radiation initiate an inflammatory process that can increase the risk of developing CVD. Inflammation initiates atherosclerosis via immune cell infiltration subsequent to endothelial cell damage. We hypothesized that CD14+ monocytes will exhibit an amplified inflammatory response, as demonstrated by significant increases in the expression of inflammatory cytokines at 48 and 72 hours post radiation exposure compared to controls which would be magnified by the inflammatory stimulus LPS. Methods: Human venous peripheral blood was collected from 5 healthy young adults (24 ± 3 years, 22 ± 1 BMI, 5 Male) using an institutional review board (IRB)-approved protocol. Women were studied in days 1-5 of menstrual cycle. Peripheral blood mononuclear cells (PBMCs) were harvested by density gradient separation and re-suspended and incubated with anti-human CD14 microbeads. CD14+ immune cells were incubated in plates, exposed to 4 gray (Gy) of x-ray radiation, and then incubated for 48 and 72 hours. CD14+ immune cells were exposed to 1:500 dilution of 0.1 mg/mL of lipopolysaccharide (LPS) for the final 8 hours of incubation. The culture media was recovered, and assayed for secreted factors, specifically for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α. Additionally, flow cytometry analysis was performed to quantify intracellular content of IL-10, IL-8, IFN-g, IL-4, receptor for advanced glycation end products (RAGE), and damage-associated molecular pattern (DAMP) molecules (S100A8/9) involved in inflammatory activation. All data were analyzed with two-way repeated measures ANOVA. Results: Secreted Cytokines: Cytokine expression at 48 and 72 hours did not differ between the control, radiation (4Gy), and 4Gy+LPS groups. At 48 and 72 hours, there were no significant differences observed in the expression levels of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α in control, 4Gy, and 4Gy+LPS cells. Intracellular Content: Compared to control IL-4 content was increased at 48 and 72 hours, after exposure to 4Gy (48h: p=0.013 and 72h: p=0.008) and 4Gy+LPS (48h: p=0.022 and 72h: p=0.002). Furthermore, IL-4 was again increased at 72 hours after exposure to 4Gy+LPS compared to 48 hours (p=0.014). IL-10 content was also increased in after exposure to 4Gy and 4Gy+LPS at 72 hours compared to control (p=0.046 and p=0.007, respectively). S100A8/9 also showed a trending increase at 4Gy compared to control at 72 hours (p=0.11). No other content changed significantly. Conclusion: These data suggest that acute radiation exposure (4 Gy x-ray) has no effect on the secretome of CD14+ immune cells. However, the intracellular content of IL-4 was increased at both 48 and 72 hours, while IL-10 increased at 72 hours, indicating a shift from pro-inflammatory to anti-inflammatory intracellular content the further in time monocytes are from radiation exposure. Future work needs to test increased and repeated doses of radiation, as well as examine both x-ray and proton exposure. TRISH through NASA Cooperative Agreement NNX16AO69A and NIH HL150361. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research.

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.

Nanoconfined Electrokinetic Chromatography (NEC): Gradient Separation and Sensing of Short DNA Fragments at the Single-Molecule Level.

Glass nanopipets have been demonstrated to be a powerful tool for the sensing and discrimination of biomolecules, such as DNA strands with different lengths or configurations. Despite progress made in nanopipet-based sensors, it remains challenging to develop effective strategies that separate and sense in one operation. In this study, we demonstrate an agarose gel-filled nanopipet that enables hyphenated length-dependent separation and electrochemical sensing of short DNA fragments based on the electrokinetic flow of DNA molecules in the nanoconfined channel at the tip of the nanopipet. This nanoconfined electrokinetic chromatography (NEC) method is used to distinguish the mixture of DNA strands without labels, and the ionic current signals measured in real time show that the mixed DNA strands pass through the tip hole in order according to the molecular weight. With NEC, gradient separation and electrochemical measurement of biomolecules can be achieved simultaneously at the single-molecule level, which is further applied for programmable gene delivery into single living cells. Overall, NEC provides a multipurpose platform integrating separation, sensing, single-cell delivery, and manipulation, which may bring new insights into advanced bioapplication.

Drag reduction in a spanwise-infinite boat-tailed body through multiple transverse grooves

Appropriately-designed spanwise-extruded small grooves, oriented transverse to the freestream flow, may delay flow separation in adverse pressure gradients by generating local and stable recirculation zones. In this study, we explore the potential of multiple grooves to reduce the drag on a boat-tailed bluff body characterized by vortex shedding. Numerical simulations reveal that the presence of two consecutive transverse grooves results in a maximum boat-tail drag reduction of 23.2%, further decreasing the drag by 15% when compared to the performance of the same boat-tailed bluff body with a single groove. This flow-control mechanism proves effective when the flow reattachment takes place downstream of both grooves throughout the entire vortex-shedding cycle, leading to the formation of two distinct local recirculation zones within the grooves. Lower frictional losses are observed along the streamline bounding the recirculation within the grooves as compared to the friction losses caused by the no-slip boundary conditions along the lateral walls of a plain boat tail (i.e., without grooves). Consequently, the boundary layer exhibits higher momentum downstream of the grooves, resulting in a delayed flow separation and an improved reduction in body drag for the boat tail.

SAP-expressing T peripheral helper cells identify systemic lupus erythematosus patients with lupus nephritis.

T follicular (TFH) and peripheral helper (TPH) cells have been increasingly recognized as a pathogenic subset of CD4 T cells in systemic lupus erythematosus (SLE). The SLAM Associated Protein (SAP) regulates TFH and TPH function by binding to the co-stimulatory signaling lymphocyte activation molecule family (SLAMF) receptors that mediate T cell - B cell interactions. SAP and SLAMF are critical for TPH-dependent B cell maturation into autoantibody-producing plasma cells that characterize SLE pathogenesis. We hypothesized that SAP-expressing TPH cells are involved in the pathogenesis of lupus nephritis (LN). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient separation from whole blood. Cells were stained for cell surface markers, followed by permeabilization and staining of intracellular SAP for spectral flow cytometry analysis. We also analyzed SAP expression from renal infiltrating LN T cells using the available single-cell RNA sequencing (scRNA seq) Accelerated Medicines Partnership (AMP) SLE dataset. PBMC from 30 patients with SLE (34 ± 10 years old, 83% female), including 10 patients with LN, were analyzed. We found an increase in total SAP-positive CD4 and CD8 T cells in SLE compared with controls (55.5 ± 2.6 vs. 41.3 ± 3.4, p=0.007, and 52.5 ± 3.0 vs. 39.2 ± 2.8, p=0.007 respectively). In CD4 T cells, the highest SAP expression was in the TPH subset. The frequency of SAP+TPH in circulation correlated with disease activity; SLE patients with renal disease had higher levels of circulating SAP+TPH that remained significant after adjusting for age, sex, race, low complements, and elevated anti-dsDNA (p=0.014). scRNA-seq data of renal infiltrating T cells in LN identified SAP expression to localize to the TFH-like CD4 cluster and GZMK+ CD8 cluster. Increased SAP expression in LN was associated with the differential expression of SLAMF3 and SLAMF7 and granzyme K and EOMES. The existence of two predominant SAP-expressing subsets, the TFH-like CD4 T cells, and GZMK+ effector CD8 T cells, was verified using scRNA-seq data from a human transcriptomic atlas of fifteen major organs. The expansion of SAP-expressing T helper cells was associated with LN in our cohort and verified using scRNA-seq data of renal infiltrating T cells. Improved SLAM and SAP signaling understanding can identify new therapeutic targets in LN.

An evaluation of microplastic contamination in the marine waters and species in the coastal region of the South Yellow Sea, China

Microplastics (MPs) contamination of marine environments poses a significant ecological risk, although impacts on species’ realized niche spaces remain unclear. The current study investigates MPs distribution across pelagic habitats, benthic sediments, and key biota in the South Yellow Sea, China. Samples were collected via trawling across estuarine transects, and tissues were digested to extract MPs. Density gradient separations and vacuum-filtrations prepared particle extracts for ATR-FTIR and Micro-Raman spectroscopic characterization. Sampling along industrialized river transects reveals ubiquitous plastic particle presence, with concentrations ranging from 0 to 51.68 item/L seawater. Contamination levels reach their peak at station estuaries before dispersing offshore, indicating significant waste stream inputs. Importantly, MPs detected in demersal and pelagic fish species, as well as in bivalves, confirm exposure across trophic niches. Gastrointestinal tract and gill concentrations reached 0.6 items/g fresh tissue, reflecting significant biological uptake and in vivo retention. The greatest population of organisms occurred adjacent to polluted areas. Overall, distribution of MPs from polluted rivers to coastal food webs was evident, suggesting potential negative impacts on key ecological functions in this system. These findings underscore the need to develop upstream mitigation efforts so as to minimize MPs contamination in areas where nearshore and offshore niches intersect.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Digital light processing 3D printing of microfluidic devices targeting high-pressure liquid-phase separations.

This paper focuses on 3D printing using digital light processing (DLP) to create microchannel devices with inner diameters of100, 200, and 500 µm and cater flow-through applications within the realm of analytical chemistry, in particular high-pressure liquid chromatographic separations. Effects of layer thickness and exposure time on channel dimensions and surface roughness were systematically investigated. Utilizing a commercially accessible 3D printer and acrylate resin formulation, we fabricated 100-500 µm i.d. squared and circular channel designs minimizing average surface roughness (< 20%) by applying a 20-µm layer thickness and exposure times ranging from 1.1 to 0.7s. Pressure resistance was measured by encasing microdevices in an aluminum chip holder that integrated flat-bottom polyetheretherketon (PEEK)nanoports allowing to establish the micro-to-macro interface to the HPLC instrument. After thermal post-curing and finetuning the clamping force of the chip holder, a maximum pressure resistance of 650bar (1.5% RSD) was reached (n = 3). A polymer monolithic support structure was successfully synthesized in situ with the confines of a 500 µm i.d. 3D printed microchannel. A proof-of-concept of a reversed-phase chromatographic gradient separation of intact proteins is demonstrated using an aqueous-organic mobile-phase with isopropanol as organic modifier.

Non-graphitic carbon as sodium-ion battery anode materials with improved ratio of plateau to sloping capacities prepared from lignite-based organic maceral

Herein, huminite concentrate is obtained from lignite with high yield of 68.9 % by density gradient separation, subsequently converted to non-graphitic carbon (HM-H) by carbonization at 1400 °C for 2 h. Huminite concentrate possesses relatively high oxygen content and considerable amount of aromatic component, with modest yield of volatile matter during pyrolysis, resulting in optimized carbon phase distribution and enhanced electrical conductivity of HM-H. Compared with HM prepared by direct carbonization of lignite, HM-H has slightly higher contents of pseudo-graphitic and graphite-like carbons, and lower content of highly disordered one. Notably, HM-H exhibits remarkably improved electrical conductivity (1773 S m−1) than HM (1406 S m−1). Compared with HM anode for SIBs, HM-H one delivers higher plateau capacity (170.1 mAh g−1) meanwhile lower sloping capacity (91.8 mAh g−1), with significantly improved rate and cycling performances. Full cell assorted with NaFe1/3Ni1/3Mn1/3O2 cathode and HM-H anode demonstrates higher energy density of 210.5 Wh kg−1 than its counterpart (193.8 Wh kg−1) at 20 mA g−1, with superior rate performance (208.6 mAh g−1 at 200 mA g−1) and a capacity retention ratio of 89.7 % after 100 cycles. This work provides a facile and effective strategy for preparing non-graphitic carbon as high-performance anode materials for SIBs.