Plasma Activation of Microplates Optimized for One-Step Reagent-Free Immobilization of DNA and Protein.

Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3-4) and streptavidin (PAC, 21% nitrogen, pH 5-7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation.

Covalent Immobilization of N-Acetylcysteine on a Polyvinyl Chloride Substrate Prevents Bacterial Adhesion and Biofilm Formation.

Biofilm formation and antimicrobial resistance at surgical implant sites result in high morbidity and mortality. Identifying novel molecules that inhibit biofilm formation to coat surgical biomaterials is essential. One such compound is N-acetylcysteine (NAC), a potent antioxidant precursor for glutathione, necessary in mammalian cells and known to disrupt/prevent biofilms. In this study, NAC was covalently immobilized onto functionalized polyvinyl chloride surfaces using plasma immersion ion implantation (PIII) treatment that achieves covalent binding without the need for linker groups. NAC immobilization was characterized using water contact angles, Fourier-transform infrared, and X-ray photoelectron spectroscopy techniques. Bacterial viability and biofilm formation on NAC surfaces were assessed using resazurin assays, phase contrast microscopy, and colony counting experiments. Effect of NAC on bacterial polysaccharide production and DNA cleaving was investigated using the phenol-sulfuric acid method and the Qubit fluorometer. Surface thermodynamics between the NAC coating and bacterial cells were measured using the Lewis acid-base method. Surface characterization techniques demonstrated superficial changes after PIII treatment and subsequent covalent NAC immobilization. NAC-coated surfaces significantly reduced biofilm viability and the presence of Gram-negative and Gram-positive bacteria. NAC also decreased polysaccharide production and degraded DNA. This led to unfavorable conditions for biofilm formation on NAC-coated surfaces, as demonstrated by surface thermodynamic analysis. NAC-coated surfaces showed no cytotoxicity to human fibroblast cells. This study has successfully utilized NAC as an antibiofilm coating, which may pave the way for improved prophylactic coatings on medical implant devices in the future.

Linker-protein G mediated functionalization of polystyrene-encapsulated upconversion nanoparticles for rapid gene assay using convective PCR.

The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 103 copies µL-1. The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 107 and 103 copies of DNA. Graphical abstract Schematic representation of polystyrene-encapsulated upconversion nanoparticles (PS-UCNPs) prepared by mini-emulsion polymerisation. The PS-UCNPs were functionalised with anti-digoxigenin antibody using the fusion protein linker-protein G (LPG). Detection of digoxigenin-labelled amplicons is achieved (a) by using the antibody-functionalised LPG@PS-UCNP labels; (b) magnetic separation, and (c) 980 nm laser light for detection of the green upconversion luminescence peaking at 537 nm.

Collaboration between health sciences librarians and faculty as reflected by articles published in the Journal of the Medical Library Association.

A recent study by Higgins and colleagues reports that the Journal of the Medical Library Association (JMLA) had the highest percentage of articles with both librarian and faculty coauthors out of 13 peer-reviewed journals in science, technology, engineering, and medicine librarianship and education between 2005 and 2014. A deeper and updated analysis of JMLA research articles and case studies published between 2008 and 2017 revealed that 29% of articles had both librarian and faculty coauthors. The main topics of librarian-faculty collaboration, as described in these articles, were related to patient and consumer health information and clinical information-seeking and decision-making by health care providers. Most faculty coauthors came from the disciplines of biomedical or health informatics and biostatistics and library and information science. The publication of these articles in the JMLA provides evidence of health sciences librarians' and information specialists' ability to collaborate with faculty members to advance the knowledgebase and practice of librarianship and the health sciences.

Contribution of carbon footprint research towards the triple bottom line of sustainability.

Carbon footprint (CF) research has received increasing attention in recent years, as evidenced by a rise in publications and citations, reflecting a growing concern for the environmental impact of human activities. However, the alignment of this scientific literature with the three dimensions of sustainability performance provided by the TBL paradigm (people, planet, and profit) has received limited attention. This study addresses this research gap by undertaking a large-scale bibliometric analysis of 9032 Web of Science (WoS) publications from 1992 to 2020. At the macro (journals) and micro (papers) levels, a methodology approach to classify research publications according to TBL dimensions was designed. The results indicate that the output and impact of CF research are balanced with respect to the environmental (planet) and economic (prosperity/profit) dimensions, while the social impact is balanced with respect to the people+profit dimensions. Other than that, "Affordable and Clean Energy" (3761 publications) and "Climate Action" (3091 publications) are the most frequently represented (and interconnected) objectives. The results obtained contribute to a greater understanding of the contribution of CF research to the attainment of the SDGs.

Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion.

A developing understanding suggests that spatial compartmentalisation in pancreatic ß cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact ß-cell structure, and enhances glucose-dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating ß cells. Integrins and FAK are exclusively activated at the ß-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where ß-cell contact the islet capillaries.

Biomaterial Wettability Affects Fibrin Clot Structure and Fibrinolysis.

Thrombosis on blood-contacting medical devices can cause patient fatalities through device failure and unstable thrombi causing embolism. The effect of material wettability on fibrin network formation, structure, and stability is poorly understood. Under static conditions, fibrin fiber network volume and density increase in clots formed on hydrophilic compared to hydrophobic polystyrene surfaces. This correlates with reduced plasma clotting time and increased factor XIIa (FXIIa) activity. These structural differences are consistent up to 50 µm away from the material surface and are FXIIa dependent. Fibrin forms fibers immediately at the material interface on hydrophilic surfaces but are incompletely formed in the first 5 µm above hydrophobic surfaces. Additionally, fibrin clots on hydrophobic surfaces have increased susceptibility to fibrinolysis compared to clots formed on hydrophilic surfaces. Under low-flow conditions, clots are still denser on hydrophilic surfaces, but only 5 µm above the surface, showing the combined effect of the surface wettability and coagulation factor dilution with low flow. Overall, wettability affects fibrin fiber formation at material interfaces, which leads to differences in bulk fibrin clot density and susceptibility to fibrinolysis. These findings have implications for thrombus formed in stagnant or low-flow regions of medical devices and the design of nonthrombogenic materials.

Single Step Plasma Process for Covalent Binding of Antimicrobial Peptides on Catheters To Suppress Bacterial Adhesion.

Catheter-associated biofilms are responsible for a large fraction of hospital acquired infections. Antimicrobial surface coating on catheters providing prevention at source is extensively studied to reduce bacterial adhesion. Antimicrobial peptides such as melimine and Mel4, covalently linked to surfaces, have shown excellent potential in animal and human studies to suppress infection without toxicity. Covalent binding of the peptides on catheter surfaces improves efficacy but so far has been implemented using multistep wet chemical coupling that will impede widespread adoption. Here we demonstrate plasma immersion ion implantation (PIII) as a single step treatment that covalently couples antimicrobial peptides to polyvinyl chloride (PVC). Strong antimicrobial activity was demonstrated by higher than 3 log kill of S. aureus. A variant of the process was demonstrated as an antimicrobial treatment for chemically inert glass surfaces. Covalent coupling was rigorously tested by stringent SDS washing. We further demonstrated that the plasma treatment can effectively functionalize both internal and external surfaces of catheter tubing, reducing 99% of bacterial adhesion. The process is feasible as a patient-safe treatment for treating various types of catheters and is suitable for commercial mass production. In a logical extension of the work, the process could be adapted to bone replacement scaffolds of all types including metallic, polymeric, and ceramic.

A plasma ion bombardment process enabling reagent-free covalent binding of multiple functional molecules onto magnetic particles.

We report a plasma immersion ion implantation process for functionalizing polymer coated magnetic particles, converting them into a universal covalent binding platform for the simultaneous binding of multiple molecular agents without the need for specialised chemical linking groups. As an example, we demonstrate the improvement of wettability and the control of surface charge of polystyrene coated magnetic particles, enhancing biomolecule attachment density with strong covalent binding. We demonstrate the preparation of multifunctional magnetic particles where two or more types of molecule are co-immobilized. This enables a platform technology with simultaneous multiple covalent binding of molecules drawn from oligonucleotides, antibodies and enzymes suitable for targeted nanoparticle diagnostic and therapies.

Increasing binding density of yeast cells by control of surface charge with allylamine grafting to ion modified polymer surfaces.

Plasma immersion ion implantation (PIII) treatment of polymers creates a biointerface capable of direct covalent immobilization of biomolecules. The immobilization of protein molecules is achieved by covalent bonds formed between embedded radicals on the treated surface and amino acid side chains and cells can be immobilized through cell-wall proteins. The attachment density of negatively charged entities on a PIII treated surface is inhibited by its negative surface charge at neutral pH. To reduce the negative charge of PIII treated surfaces in phosphate buffer (pH 7.4, 11mM), we develop an effective approach of grafting allylamine monomers onto the treated surface. The results reveal reactions between allylamine and radicals on the PIII treated surface. One of these triggers polymerization, increasing the number of amine groups grafted. As a consequence, the PIII treated polystyrene surface after allylamine exposure becomes more hydrophobic and less negatively charged in phosphate buffer. Using yeast cells as an example, we have shown a significant improvement (6-15 times) of cell density immobilized on the PIII treated surface after exposure to allylamine.

Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.

Plasma immersion ion implantation (PIII) treatment is a novel method for immobilizing yeast on polymer surfaces by covalent linkage. This study of the immobilization of Saccharomyces cerevisiae in both rehydrated and cultured forms showed that the density of cell attachment on PIII treated polystyrene (PS) was strongly dependent on the pH of the incubation medium and was higher for rehydrated yeast. A study of the surface charge was undertaken to explain this result. A high density of cell attachment occurs in acidic conditions (pH 3-5) and a significantly reduced cell density occurs in neutral and alkaline buffers (pH 6-10) for both types of yeast. Force measurements using atomic force microscopy show that a negative charge is present on polystyrene after PIII treatment. The charge is close to zero at pH 3 to pH 5 and increasingly negative from pH 6 to pH 10. Both rehydrated yeast and cultured yeast have negative electrophoretic mobility in the pH range studied. The repulsive forces are weak in acidic buffers and stronger in neutral and alkaline buffers, in good agreement with the cell densities observed. Rehydrated yeast cells are found to be more hydrophobic than cultured yeasts in the same buffer. The higher hydrophobicity explains the higher attachment of rehydrated yeast compared to cultured yeast.

Ion-implanted polytetrafluoroethylene enhances Saccharomyces cerevisiae biofilm formation for improved immobilization.

The surface of polytetrafluoroethylene (PTFE) was modified using plasma immersion ion implantation (PIII) with the aim of improving its ability to immobilize yeast. The density of immobilized cells on PIII-treated and -untreated PTFE was compared as a function of incubation time over 24 h. Rehydrated yeast cells attached to the PIII-treated PTFE surface more rapidly, with higher density, and greater attachment strength than on the untreated surface. The immobilized yeast cells were removed mechanically or chemically with sodium hydroxide and the residues left on the surfaces were analysed with Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). The results revealed that the mechanism of cell attachment on both surfaces differs and a model is presented for each. Rapid attachment on the PIII-treated surface occurs through covalent bonds of cell wall proteins and the radicals on the treated surface. In contrast, on the untreated surface, only physisorbed molecules were found in the residue and lipids were more highly concentrated than proteins. The presence of lipids in the residue was found to be a consequence of damage to the plasma membrane during the rehydration process and the increased cell stress was also apparent by the amount of Hsp12 in the protein residue. The immobilized yeast cells on PIII-treated PTFE were found to be as active as yeast cells in suspension.