Synergistic antibacterial attributes of copper-doped polydopamine nanoparticles: an insight into photothermal enhanced antibacterial efficacy.

Antibiotic-resistant bacteria and associated infectious diseases pose a grave threat to human health. The antibacterial activity of metal nanoparticles has been extensively utilized in several biomedical applications, showing that they can effectively inhibit the growth of various bacteria. In this research, copper-doped polydopamine nanoparticles (Cu@PDA NPs) were synthesized through an economical process employing deionized water and ethanol as a solvent. By harnessing the high photothermal conversion efficiency of polydopamine nanoparticles (PDA NPs) and the inherent antibacterial attributes of copper ions, we engineered nanoparticles with enhanced antibacterial characteristics. Cu@PDA NPs exhibited a rougher surface and a higher zeta potential in comparison to PDA NPs, and both demonstrated remarkable photothermal conversion efficiency. Comprehensive antibacterial evaluations substantiated the superior efficacy of Cu@PDA NPs attributable to their copper content. These readily prepared nano-antibacterial materials exhibit substantial potential in infection prevention and treatment, owing to their synergistic combination of photothermal and spectral antibacterial features.

Inducing Intermediates in Biotransformation of Natural Polyacetylene and A Novel Spiro-γ-Lactone from Red Ginseng by Solid Co-Culture of Two Gut Chaetomium globosum and The Potential Bioactivity Modification by Oxidative Metabolism.

The ω-hydroxyl-panaxytriol (1) and ω-hydroxyl-dihydropanaxytriol (2)-are rare examples of polyacetylene metabolism by microbial transformation, and these new metabolites (1, 2) from fermented red ginseng (FRG) by solid co-culture induction of two Chaetomium globosum should be the intermediates of biotransformation of panaxylactone (metabolite A). The metabolic pathway of panaxylactone was also exhibited. The ingredients of red ginseng (RG) also induced the production of rare 6/5/5 tricyclic ring spiro-γ-lactone skeleton (3). The ω-hydroxylation of new intermediates (1, 2) decreases cytotoxicity and antifungal activity against C. globosum compared with that of its bioprecursor panaxytriol. Additionally, compounds 1 and 2 indicated obvious inhibition against nitric oxide (NO) production, with ratios of 44.80 ± 1.37 and 23.10 ± 1.00% at 50 µM. 1 has an equivalent inhibition of NO production compared with the positive drug. So, the microbial biotransformation that occurred in FRG fermented by gut C. globosum can change the original bioactivity of polyacetylene, which gave a basis about the metabolic modification of red ginseng by intestinal fungus fermentation.

Polyethylene glycol loxenatide (PEX168) in subjects with renal impairment: A pharmacokinetic study.

AIMS: Type 2 diabetes mellitus (T2DM) is commonly complicated by renal impairment. Polyethylene glycol loxenatide (PEX168) is a novel long-acting glucagon-like peptide-1 receptor agonist for T2DM. PEX168 pharmacokinetics was studied to identify requirements for dose-modification in T2DM complicated by renal impairment. METHODS: This was a single-centre, open-labelled, parallel-group, single-dose, phase I clinical trial of patients with mild and moderate renal impairment, and with or without T2DM. Age-, sex- and body mass index-matched subjects with normal renal function, and with or without T2DM were recruited as controls. Subjects received a single abdominal subcutaneous injection of PEX168 200 µg. Pharmacokinetic samples were taken at 0, 24, 48, 72, 96, 120, 144, 216, 312, 480, 648 and 720 hours. RESULTS: Twenty-three patients were included in the pharmacokinetics analysis. Vz/F and CL/F were lower in the moderate impairment group than in the other groups. The mean t1/2 (163 hours) in the moderate impairment group was prolonged compared to the mild impairment (117 hours) and normal (121 hours) groups. AUC0-inf increased by 13 and 100.7% in patients with mild and moderate renal impairment, respectively. Most adverse events were mild gastrointestinal disorders, with only 1 serious adverse event observed. CONCLUSION: A single dose of 200 µg of PEX168 was in general well tolerated in patients with renal impairment. The in vivo clearance rate of PEX168 in patients with moderate renal impairment is slower than in patients with mild renal impairment and normal renal function and dose adjustment might be required (ClinicalTrials.org #NCT02467790).

Membrane rigidity induced by grafted polymer brush.

The contribution of neutral polymer brush to the curvature elasticity of the grafting surface is investigated theoretically. Using self-consistent field theory, we accurately evaluate the dependence of bending modulus on parameters including chain length, Flory-Huggins parameter and grafting density and reveal the importance of solvent. The results show that the brush-induced bending modulus follows a complex dependence on grafting density and Flory-Huggins parameter, while it obeys a simple power law with chain length as N(3). The method is further applied to calculate the polymer brush's contribution to the elastic properties of PEG-grafted lipid monolayers.

Layer-by-layer coated probiotics with chitosan and liposomes demonstrate improved stability and antioxidant properties in vitro.

Probiotics are of increasing interest for their potential health benefits, but their survival and adhesion in the harsh gastrointestinal environment remain a concern. This study explored a single-cell encapsulation technique to enhance probiotic survival and adhesion in the gastrointestinal tract. We encapsulated probiotics in curcumin-loaded liposomes, further coated them with polymers using layer-by-layer techniques. The coated probiotics were evaluated for survival in simulated gastrointestinal conditions, adhesion to colonic mucus, and scavenging of reactive oxygen species (ROS). The results showed that multi-layer encapsulation increased probiotic size at the nanoscale, enhancing their survival in simulated gastrointestinal conditions. Upon reaching the colon, the shedding of the coating coincided with probiotic proliferation. Additionally, the coated probiotics exhibited increased adhesion to colonic mucus. Moreover, the coating acted as a protective barrier for effectively scavenging reactive oxygen radicals, ensuring probiotic survival in inflammatory environments. This study combines the synergistic effects of probiotics and curcumin, underscoring the promise of single-cell encapsulation techniques in improving the efficacy of probiotics for addressing colitis-related diseases.

Machine learning-based prediction of methane production from lignocellulosic wastes.

The biochemical methane potential test is a standard method to determine the biodegradability of lignocellulosic wastes (LWs) during anaerobic digestion (AD) with disadvantages of long experiment duration and high operating expense. This paper developed a machine learning model to predict the cumulative methane yield (CMY) using the data of 157 LWs regarding physicochemical characteristics, digestion condition and methane yield, with the coefficient of determination equal to 0.869. Model interpretability analyses underscored lignin content, organic loading, and nitrogen content as pivotal attributes for CMY prediction. For the feedstocks with a cellulose content exceeding about 50%, the CMY in the early AD stage would be relatively lower than those with low cellulose content, but prolonging digestion time could promote methane production. Besides, lignin content in feedstock surpassing 15% would significantly inhibit methane production. This work contributes to valuable guidance for feedstock selection and operation optimization for AD plants.

Red Blood Cell-Like Poly(ethylene glycol) Particles: Influence of Particle Stiffness on Biological Behaviors.

Cell-like particles represent a category of synthetic particles designed to emulate the structures or functions of natural cells. Herein, we present the assembly of cell-like poly(ethylene glycol) (PEG) particles with different stiffnesses and shapes via replication of animal cells and investigate the impact of particle stiffness on their biological behaviors. As a proof of concept, we fabricate red blood cell-like and spherical PEG particles with varying cross-linking densities. A systematic exploration of their properties, encompassing morphology, stiffness, deformability, and biodistribution, reveal the vital influence of particle stiffness on in vivo fate, elucidating its role in governing the traversal of capillaries and the dynamic interactions with phagocytic cells.

2-Monoacylglycerol Mimetic Liposomes to Promote Intestinal Lymphatic Transport for Improving Oral Bioavailability of Dihydroartemisinin.

Purpose: Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a primary digestive product of dietary lipids triglyceride, can be assembled in chylomicrons and then transported from the intestine into the lymphatic system. Herein, we propose a biomimetic strategy and report a 2-MAG mimetic nanocarrier to target the intestinal lymphatic system via the lipid absorption pathway and improve oral bioavailability. Methods: The 2-MAG mimetic liposomes were designed by covalently bonding serinol (SER) on the surface of liposomes named SER-LPs to simulate the structure of 2-MAG. Dihydroartemisinin (DHA) was chosen as the model drug because of its disadvantages such as poor solubility and high first-pass effect. The endocytosis and exocytosis mechanisms were investigated in Caco-2 cells and Caco-2 cell monolayers. The capacity of intestinal lymphatic transport was evaluated by ex vivo biodistribution and in vivo pharmacokinetic experiments. Results: DHA loaded SER-LPs (SER-LPs-DHA) had a particle size of 70 nm and a desirable entrapment efficiency of 93%. SER-LPs showed sustained release for DHA in the simulated gastrointestinal environment. In vitro cell studies demonstrated that the cellular uptake of SER-LPs primarily relied on the caveolae- rather than clathrin-mediated endocytosis pathway and preferred to integrate into the chylomicron assembly process through the endoplasmic reticulum/Golgi apparatus route. After oral administration, SER-LPs efficiently promoted drug accumulation in mesenteric lymphatic nodes. The oral bioavailability of DHA from SER-LPs was 10.40-fold and 1.17-fold larger than that of free DHA and unmodified liposomes at the same dose, respectively. Conclusion: SER-LPs improved oral bioavailability through efficient intestinal lymphatic transport. These findings of the current study provide a good alternative strategy for oral delivery of drugs with high first-pass hepatic metabolism.

Neural Progenitor Cell-Mediated Magnetic Nanoparticles for Magnetic Resonance Imaging and Photothermal Therapy of Glioma.

Glioma is the most common primary malignant tumor in the brain. The diagnostic accuracy and treatment efficiency of glioma are facing great challenges due to the presence of the blood-brain barrier (BBB) and the high infiltration of glioma. There is an urgent need to explore the combination of diagnostic and therapeutic approaches to achieve a more accurate diagnosis, as well as guidance before and after surgery. In this work, we induced human induction of pluripotent stem cell into neural progenitor cells (NPCs) and synthesized nanoprobes labeled with enhanced green fluorescent protein (EGFP, abbreviated as MFe3O4-labeled EGFP-NPCs) for photothermal therapy. Nanoprobes carried by NPCs can effectively penetrate the BBB and target glioma for the purpose of magnetic resonance imaging and guiding surgery. More importantly, MFe3O4-labeled EGFP-NPCs can effectively induce local photothermal therapy, conduct preoperative tumor therapy, and inhibit the recurrence of postoperative glioma. This work shows that MFe3O4-labeled EGFP-NPCs is a promising nanoplatform for glioma diagnosis, accurate imaging-guided surgery, and effective photothermal therapy.

Critical conditions of polymer chromatography: an insight from SCFT modeling.

In polymer chromatography, chain molecules are separated by molecular weight, size, and chemical composition due to adsorption and exclusion in nanoporous substrates. Three regimes of separation are distinguished depending on the adsorption strength and the pore size. In the regime of size exclusion chromatography, the adsorption energy is weak and the separation is entropy-driven with larger molecules having shorter retention times. On the opposite, in the regime of adsorption chromatography, enthalpy gain due to strong adsorption energy prevails over entropy loss, and the retention time of smaller molecules is shorter. We study the intermediate regime of so-called critical conditions, at which the entropic and enthalpic effects are mutually compensated, and the partition coefficient does not depend on the polymer molecular weight. Using the self-consistent field theory of tethered polymer chains, we confirm that for ideal chains the critical conditions are justified, albeit they depend on the pore size. However, for real chains with the excluded volume effect, the critical conditions hold only approximately, and the discrepancy increases as the pore size decreases. We show that it is important to consider three characteristic adsorption states: chains adsorbed at the external surface, chains adsorbed completely inside the pores, and partially translocated chains or "flowers" with a "root" adsorbed inside the pore and a "stem" hanging outside. The interplay of different adsorption mechanisms and the pore size distribution inherent to real substrates may lead to the manifestation of apparent critical adsorption conditions within the inherent deviation of experimental data.

An Optimized Method for Microcomputed Tomography Analysis of Trabecular Parameters of Metal Scaffolds for Bone Ingrowth.

Owing to its superior mechanical and biological properties, titanium metal is widely used in dental implants, orthopedic devices, and bone regenerative materials. Advances in 3D printing technology have led to more and more metal-based scaffolds being used in orthopedic applications. Microcomputed tomography (µCT) is commonly applied to evaluate the newly formed bone tissues and scaffold integration in animal studies. However, the presence of metal artifacts dramatically hinders the accuracy of µCT analysis of new bone formation. To acquire reliable and accurate µCT results that reflect new bone formation in vivo, it is crucial to lessen the impact of metal artifacts. Herein, an optimized procedure for calibrating µCT parameters using histological data was developed. In this study, the porous titanium scaffolds were fabricated by powder bed fusion based on computer-aided design. These scaffolds were implanted in femur defects created in New Zealand rabbits. After 8 weeks, tissue samples were collected to assess new bone formation using µCT analysis. Resin-embedded tissue sections were then used for further histological analysis. A series of deartifact two-dimensional (2D) µCT images were obtained by setting the erosion radius and the dilation radius in the µCT analysis software (CTan) separately. To get the µCT results closer to the real value, the 2D µCT images and corresponding parameters were subsequently selected by matching the histological images in the particular region. After applying the optimized parameters, more accurate 3D images and more realistic statistical data were obtained. The results demonstrate that the newly established method of adjusting µCT parameters can effectively reduce the influence of metal artifacts on data analysis to some extent. For further validation, other metal materials should be analyzed using the process established in this study.

Adsorption-driven translocation of polymer chain into nanopores.

The polymer translocation into nanopores is generally facilitated by external driving forces, such as electric or hydrodynamic fields, to compensate for entropic restrictions imposed by the confinement. We investigate the dynamics of translocation driven by polymer adsorption to the confining walls that is relevant to chromatographic separation of macromolecules. By using the self-consistent field theory, we study the passage of a chain trough a small opening from cis to trans compartments of spherical shape with adsorption potential applied in the trans compartment. The chain transfer is modeled as the Fokker-Plank diffusion along the free energy landscape of the translocation pass represented as a sum of the free energies of cis and trans parts of the chain tethered to the pore opening. We investigate how the chain length, the size of trans compartment, the magnitude of adsorption potential, and the extent of excluded volume interactions affect the translocation time and its distribution. Interplay of these factors brings about a variety of different translocation regimes. We show that excluded volume interactions within a certain range of adsorption potentials can cause a local minimum on the free energy landscape, which is absent for ideal chains. The adsorption potential always leads to the decrease of the free energy barrier, increasing the probability of successful translocation. However, the translocation time depends non-monotonically of the magnitude of adsorption potential. Our calculations predict the existence of the critical magnitude of adsorption potential, which separates favorable and unfavorable regimes of translocation.

Cellulose or chitin nanofibril-stabilized latex for medical adhesion via tailoring colloidal interactions.

The objective of this research is to develop a functional medical adhesive from natural nanofibril-stabilized latex through an aqueous process. Surface charged cellulose or chitin nanofibrils are used to form Pickering emulsions of acrylic monomers, followed by in situ polymerization. Charged initiators are selected to tailor the interactions between them and nanofibrils, and it is found that the repulsive electrostatic interactions play a key role in stabilizing the heterogeneous system. As a result, poly(2-ethylhexyl acrylate-co-methyl methacrylate) latexes are successfully prepared for surfactant-free adhesives with a high shear strength of 72.0 ± 6.5 kPa. In addition, drug can be easily incorporated in the nanopaper substrate or adhesive layer to form a medical tape, exhibiting long-term drug release and antibacterial behaviors. We managed developing a facile method to integrate green synthesis, versatile functionalities and excellent adhesion into one adhesive, which remains a great challenge.

Screening of periodontitis-related diagnostic biomarkers based on weighted gene correlation network analysis and machine algorithms.

BACKGROUND: Periodontitis is a common oral immune inflammatory disease and early detection plays an important role in its prevention and progression. However, there are no accurate biomarkers for early diagnosis. OBJECTIVE: This study screened periodontitis-related diagnostic biomarkers based on weighted gene correlation network analysis and machine algorithms. METHODS: Transcriptome data and sample information of periodontitis and normal samples were obtained from the Gene Expression Omnibus (GEO) database, and key genes of disease-related modules were obtained by bioinformatics. The key genes were subjected to Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and 5 machine algorithms: Logistic Regression (LR), Random Forest (RF), Gradient Boosting Decisio Tree (GBDT), Extreme Gradient Boosting (XGBoost), and Support Vector Machine (SVM). Expression and correlation analysis were performed after screening the optimal model and diagnostic biomarkers. RESULTS: A total of 47 candidate genes were obtained, and the LR model had the best diagnostic efficiency. The COL15A1, ICAM2, SLC15A2, and PIP5K1B were diagnostic biomarkers for periodontitis, and all of which were upregulated in periodontitis samples. In addition, the high expression of periodontitis biomarkers promotes positive function with immune cells. CONCLUSION: COL15A1, ICAM2, SLC15A2 and PIP5K1B are potential diagnostic biomarkers of periodontitis.

Bioavailability and Safety of a New Highly Concentrated Midazolam Nasal Spray Compared to Buccal and Intravenous Midazolam Treatment in Chinese Healthy Volunteers.

INTRODUCTION: Buccal midazolam treatment is licensed in the European Union for prolonged acute convulsive seizures in children and adolescents, but the buccal pathway is often hampered by jaw clenching, hypersalivation, or uncontrolled swallowing. Midazolam formulations that are more secure, reliable, and faster for use are needed in the acute setting. Pharmacokinetics and comparative bioavailability of intranasally administered midazolam and two midazolam intravenous solutions administered buccally or intravenously in healthy adults were evaluated. METHODS: In this phase 1, open-label, randomized, single-dose, three-period, three-sequence crossover study, 12 healthy adults (19-41 years) were randomly assigned to receive 2.5 mg midazolam intranasally; 2.5 mg midazolam intravenously; 2.5 mg midazolam buccally. Blood samples were collected for 10 h post dose to determine pharmacokinetic profiles. Adverse events and vital signs were recorded. RESULTS: Intranasal administration of 2.5 mg midazolam demonstrated a more rapid median time to Cmax compared to buccal administration of midazolam (Tmax, 12.6 min vs. 45 min; Cmax, 38.33 ng/ml vs. 24.97 ng/ml). The antiepileptic effect of intranasal and buccal midazolam treatment lasted less than 4 h and generally did not differ from intravenously administered midazolam. No serious adverse events or deaths were reported, and no treatment-emergent adverse events led to study discontinuation. CONCLUSION: Intranasal administration of midazolam may be a preferable alternative to the currently approve buccal midazolam treatment for prolonged acute convulsive seizures in children and adolescents. TRIAL REGISTRATION: This study is registered at the Chinese Clinical Trial [ http://www.chictr.org.cn ] (ChiCTR2000032595) on 3 May, 2020.

cRGD peptide-conjugated polyethylenimine-based lipid nanoparticle for intracellular delivery of siRNA in hepatocarcinoma therapy.

The effective delivery system plays an important role in the application of siRNA in the antitumor study. However, until now, researches on the delivery systems targeting hepatocarcinoma cells are still being explored. Here we designed and prepared a novel siRNA delivery system, cRGD-PSH-NP, which was based on a modified polyethyleneimine (PSH) and DSPE-PEG2000-cRGD. cRGD-PSH-NP loaded with survivin siRNA (cRGD-PSH-NP/S) was composed of egg phosphatidylcholine, cationic PSH, PEGylated lipids, survivin siRNA, and cRGD peptide as a targeting ligand. The formulations of cRGD-PSH-NP/S were optimized and characterized. In vitro investigations showed excellent gene silencing and antitumor activity compared with the unmodified nanoparticles in HepG2 cells. In vivo antitumor efficacy of cRGD-PSH-NP/S exhibited potent tumor inhibition (74.71%) in HepG2-bearing nude mice without inducing toxicity. These data suggested further research of cRGD-PSH-NP/S in hepatocarcinoma therapy.

Multifunctional Ca-Zn-Si-based micro-nano spheres with anti-infective, anti-inflammatory, and dentin regenerative properties for pulp capping application.

While pulp capping using a variety of materials has been applied clinically to preserve the health and vitality of the dental pulp and induce dentin repair no material meets all the anti-infection, anti-inflammation, and promoting pulp tissue regeneration criteria. Micro-nano materials of bioactive glasses (BG) with the biocompatibility and osteogenesis-promoting properties were developed for this study using Zn-doped bioactive glass (BGz) micro-nano spheres for dental pulp capping to control infection and inflammation and promote tissue regeneration. Of three key findings, the co-culture of Porphyromonas gingivalis showed that the BGz had an excellent antibacterial effect, and after being stimulated with BGz in vitro, macrophages showed a significant decrease of pro-inflammatory M1 markers compared with the undoped BG group. It is also noted that the conditioned medium derived from BGz-stimulated macrophages could significantly promote mineralized dentin formation of dental pulp cells (DPCs). In rats, acute pulp restoration experiments proved that BGz used as a pulp capping agent had excellent dentin regenerative properties. This work may provide a novel strategy to promote osteo/dentinogenic differentiation through regulating early inflammation, with potential applications in pulp capping.

Nanostructured Zirconia Surfaces Regulate Human Gingival Fibroblasts Behavior Through Differential Modulation of Macrophage Polarization.

Zirconia exhibits excellent biocompatibility and is widely used as dental implant materials in prosthodontics. Over the past years, research and development of dental implant biomaterials has focused on osseointegration, but few reports exist regarding the role of the immune environment on cellular responses to these materials. The present study investigates the effect of different nanostructured zirconia surface topographies on macrophage phenotypes and their influence on gingival fibroblast behavior. Three different nanostructured zirconia surfaces are characterized using scanning electron microscopy, atomic force microscopy, and water contact angle. Blank-machined zirconia (BMZ) surfaces were superior to RAW264.7 cell proliferation and adhesion. RAW264.7 seeded on all nanostructured zirconia surfaces polarized toward both inflammatory M1 and anti-inflammatory M2 macrophages with more M2 macrophage phenotype on BMZ surfaces. Meanwhile, conditioned media (CM) from RAW264.7 culture on three nanostructured zirconia surfaces inhibited cell apoptosis to human gingival fibroblasts (HGFs) but promoted HGF proliferation and secretion. Under modulation of RAW264.7 culture, HGFs cultured on BMZ surfaces significantly secreted more extracellular matrix with a higher expression of collagen-I (COL-I), vinculin (VCL), and fibronectin (FN) than those coated on self-glazed zirconia (CSGZ) and self-glazed zirconia (SGZ) surfaces. After being coated with a nano zirconia film, CSGZ surfaces showed certain changes in cell proliferation, adhesion, and protein production compared with SGZ surfaces. These findings will provide an overview of manipulating surface topography to modulate macrophage phenotypes in order to create an effective macrophage immune response and reinforce soft tissue integration.

Multifunctional Chitosan/Polycaprolactone Nanofiber Scaffolds with Varied Dual-Drug Release for Wound-Healing Applications.

Electrospinning-based wound dressings with multifunctional properties, including hemostasis-promoting, antibacterial, drug release, and therapeutic effects, are of great interest in military and civilian trauma healthcare. Herein, we designed lidocaine hydrochloride (LID) and mupirocin-loaded chitosan/polycaprolactone (CSLD-PCLM) scaffolds with multiple functions as wound dressings. Through the dual spinneret electrospinning technique, the scaffolds achieved a nanofiber structure, which enhanced the interfacial interaction between the scaffold and blood cells and showed excellent blood coagulation capacity. In particular, the scaffolds loaded with LID and mupirocin exhibited rapid release of LID and sustained release of mupirocin. The CSLD-PCLM scaffold containing mupirocin exhibited outstanding antibacterial activity. Moreover, the scaffold significantly enhanced the wound healing process with complete re-epithelialization as well as collagen deposition in a full-thickness skin defect model. Thus, CSLD-PCLM nanofibrous scaffolds may ideally meet the various requirements of the wound healing process and are promising candidates for wound dressings in future clinical applications.

Microfluidic 2-D PAGE using multifunctional in situ polyacrylamide gels and discontinuous buffers.

A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS-protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design.