Attachment of bacterial cells to carbon electrodes

Anodic stripping method was applied to analyze the process of bacterial attachment to the surface of carbon-paste electrodes (CPE). The electrode was immersed for various times in a bacterial cell suspension to allow the cells to attach to its surface. The number of bacterial cells attached to the electrode surface increased along with time. On the other hand, the current derived from the oxidation of a dye, Hoechst, which was adsorbed to the surface after attaching the bacterial cells, decreased along with time. It was considered that the current output, correlated with the amount of dye, adsorbed onto regions where no bacterial cell attached. These results indicate that the bacterial-attachment process can be analyzed by measuring the electric current derived from the dye instead of counting the number of attached cells.     

Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping

A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 microm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.     

Electrochemical attachment of motile bacterial cells to gold

Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750 mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays.     

Initial bacterial attachment in slow flowing systems: effects of cell and substrate surface properties

Bacterial biofilm can have significant effects on the behaviors and/or performance of natural and man-made systems. Understanding the factors governing initial bacterial attachment is critical to biofilm management. In this study, the initial attachment of three bacteria, Pseudomonas aeruginosa, Escherichia coli and Pseudomonas putida, on two substrates, glass and octadecyltrichlorosilane (OTS) modified glass, was examined in flow chambers. The flow chambers were designed and operated to mimic slow moving water bodies and minimize the gravitational settlement of cells. The hydrophobicity of bacterial surface was evaluated by partitioning of cells to the water-hexadecane interface and the liquid contact angles on cell layers collected on filter papers. On the more hydrophilic glass surface, the attachment trend was found to be E. coli>P. putida>P. aeruginosa, while the opposite trend was observed on the hydrophobic, OTS modified surface. The attachment trend on glass could be explained by the magnitude of the negative interaction energy at secondary minima, as predicted by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. The much higher attachments of P. aeruginosa and P. putida on the OTS-modified substrate, on the other hand, suggested that these cells could overcome the energy barrier between the primary and secondary minima of interaction energy to become attached to the primary minimum. The extent of primary-minimum attachment appeared to correlate with the scale of the energy barrier, with higher attachments in the bacteria-substrate combinations of lower energy barriers. The study generated important insights into the effects of cell and substrate surface properties on initial bacterial attachment.     

Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells

In the current study, we have altered the surface oxide properties of a Ti6Al4V alloy using heat treatment or radiofrequency glow discharge (RFGD) in order to evaluate the relationship between the physico-chemical and biological properties of the alloy's surface oxide. The effects of surface pretreatments on the attachment of cells from two osteogenic cell lines (MG63 and MC3T3) and a mesenchymal stem cell line (C3H10T1/2) to fibronectin adsorbed to the alloy were measured. Both heat and RFGD pretreatments produced a several-fold increase in the number of cells that attached to fibronectin adsorbed to the alloy at a range of coating concentrations (0.001-10nM FN) for each cell line tested. An antibody (HFN7.1) directed against the central integrin binding domain of fibronectin produced a 65-70% inhibition of cell attachment to fibronectin-coated disks, indicating that cell attachment to the metal discs was dependent on fibronectin binding to cell integrin receptors. Both treatments also accelerated the cell spreading response manifested by extensive flattening and an increase in mean cellular area. The treatment-induced increases in the cell attachment activity of adsorbed fibronectin were correlated with previously demonstrated increases in Ti6Al4V oxide negative net surface charge at physiological pH produced by both heat and RFGD pretreatments. Since neither treatment increased the adsorption mass of fibronectin, these findings suggest that negatively charged surface oxide functional groups in Ti6Al4V can modulate fibronectin's integrin receptor activity by altering the adsorbed protein's conformation. Our results further suggest that negatively charged functional groups in the surface oxide can play a prominent role in the osseointegration of metallic implant materials.     

Preparation of carbohydrate arrays by using Diels-Alder reactions with inverse electron demand

Carbohydrate microarrays are an emerging tool for the high-throughput screening of carbohydrate-protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels-Alder reaction with inverse-electron-demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate-dienophile conjugates that were printed onto tetrazine-modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high-quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.     

Molecular Engineering of Surfaces by Plasma Copolymerization and Enhanced Cell Attachment and Spreading

The plasma copolymerization of acrylic acid and methyl vinyl ketone with 1,7-octadiene is reported. A range of surfaces including hydrocarbon plasma polymer, carbonyl-containing plasma copolymer and carboxyl-containing plasma copolymers have been prepared and characterized by means of X-ray photoelectron spectroscopy. The stability of these surfaces in water has been assessed at 37°C. The influence of their functional group chemistries on the attachment and spreading of osteoblast-like (ROS 17/2.8) and bone marrow stromal cells (BMSC) has been explored. The data reported show a strong correlation between the carboxyl group and the attachment and spreading of both ROS cells and BMSC. This relationship has been explored in some detail with the ROS cells. Actin-staining by direct immunofluorescence was used to visualize changes in cytoskeleton of ROS cells with substratum chemistry. As the concentration of the carboxyl groups increased cell number and cell spreading were notably enhanced. As few as 5 carboxyls per 100 carbons were sufficient to support good attachment and with cells showing well-defined polygonal cell morphology. Although cells attached to a hydrocarbon plasma polymer surface these cells had failed to spread. The attachment and spreading of BMSC were compared on a carboxyl-containing surface and on a hydrocarbon surface. There was a more marked difference in the number of cells that had attached to these two surfaces (cf. ROS cells). The spreading on the carboxyl surface was much more typical of BMSC. Results from an extended culture using primary cultures of bone marrow cells (BMC) are reported. These cells were not trypsinized and appear to be less sensitive to differences in surface chemistry (cf. BMSC). Even so, the results at 10 days indicate much greater biosynthetic activity on the carboxyl-containing surface.     

Influence of ionic strength and pH on the first 60 min of Pseudomonas aeruginosa attachment to ZnSe and to TiO2 monitored by ATR-IR spectroscopy

Establishing the factors which influence the attachment of bacteria to surfaces is important in both preventing and enhancing biofilm formation. The initial hour of attachment of Pseudomonas aeruginosa to ZnSe and to TiO₂ from solutions of different ionic strength and pH was studied using in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The TiO₂ surface was prepared by dip-coating a ZnSe internal reflection element, which produced a 50 nm thick, continuous flat film. At pH 6.3 attachment was found to increase with ionic strength up to 0.03 mol l⁻¹ but to decrease at 0.15 mol l⁻¹. At an ionic strength of 0.003 mol l⁻¹ attachment increased with pH from 4 to 6.3 to 10, but at ionic strength of 0.03 mol l⁻¹ attachment was greater at pH 6.3 than at pH 10. The influence of ionic strength appears to be due to charge factors and/or related changes in the degree of extension of bacterial surface polymers. The complex trends in the influence of pH on attachment can not be explained solely in terms of bacterial and substrate charge, bacterial surface polymer extension or bacterial metabolic activity.     

Preparation of Carbohydrate Arrays by Using Diels–Alder Reactions with Inverse Electron Demand

Carbohydrate microarrays are an emerging tool for the high‐throughput screening of carbohydrate–protein interactions that represent the basis of many biologically and medicinally relevant processes. The crucial step in the preparation of carbohydrate arrays is the attachment of carbohydrate probes to the surface. We examined the Diels–Alder reaction with inverse‐electron‐demand (DARinv) as an irreversible, chemoselective ligation reaction for that purpose. After having shown the efficiency of the DARinv in solution, we prepared a series of carbohydrate–dienophile conjugates that were printed onto tetrazine‐modified glass slides. Binding experiments with fluorescently labeled lectins proved successful and homogeneous immobilization was achieved by the DARinv. For immobilization of nonfunctionalized reducing oligosaccharides we developed a bifunctional chemoselective linker that enabled the attachment of a dienophile tag to the oligosaccharides through oxime ligation. The conjugates obtained were successfully immobilized on glass slides. The presented strategies for the immobilization of both synthetic carbohydrate derivatives and unprotected reducing oligosaccharides facilitate the preparation of high‐quality carbohydrate microarrays by means of the chemoselective DARinv. This concept can be readily adapted for the preparation of other biomolecule arrays.     

A microfluidic "baby machine" for cell synchronization

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.     

Bacterial attachment over a wide range of ionic strengths

The number of bacterial cells adhered on a glass surface was counted over a wide range of ionic strengths. The counted number increased linearly with the square root of time. The rate of attachment increased with the increase in ionic strength and then plateaued. The rate of attachment was analyzed on the basis of the potential barrier between the surface of the bacterial cell and that of the substratum. An equation for formulating the dependence of the attachment rate on the ionic strength was proposed, which seems to be useful for the systematic understanding of bacterial attachment in various environments, from terrestrial to marine.     

Bacterial attachment on reactive ceramic ultrafiltration membranes

Bacterial attachment is an initial stage in biofilm formation that leads to flux decline in membrane water filtration. This study compares bacterial attachment among three photocatalytic ceramic ultrafiltration membranes for the prevention of biofilm formation. Zirconia ceramic ultrafiltration membranes were dip-coated with anatase and mixed phase titanium dioxide photocatalysts to prevent biofilm growth. The membrane surface was characterized in terms of roughness, hydrophobicity, bacterial cell adhesion, and attached cell viability, all of which are important factors in biofilm formation. The titanium dioxide coatings had minimal impact on the membrane roughness, reduced the hydrophobicity of membranes, prevented Pseudomonas putida attachment, and reduced P. putida viability. Degussa P25 is a particularly promising reactive coating because of its ease of preparation, diminished cell attachment and viability in solutions with low and high organic carbon concentrations, and reduced flux decline. These reactive membranes offer a promising strategy for fouling resistance in water filtration systems.     

Formation and characterization of DNA microarrays at silicon nitride substrates

A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.     

Antibacterial Properties of Non-Modified Wool,Determined and Discussed in Relation to ISO 20645:2004 Standard

Wool is considered to possibly exhibit antibacterial properties due to the ability of wool clothing to reduce the build-up of odor, which arises from the microbial activity of skin microbiota. Indeed, when tested with a widely used agar diffusion plate test method, even wool or other textiles not treated with any antimicrobial agent can be interpreted to show certain antibacterial effects due to the lack of growth under the specimen, as instructed in ISO 20645:2004 standard. Therefore, we analyzed in detail what happens to bacterial cells in contact with untreated wool and cotton fabric placed on inoculated agar plates by counting viable cells attached to the specimens after 1 and 24 h of contact. All wool and several cotton samples showed no growth under the specimen. Nevertheless, it was shown without a doubt that neither textile material kills bacteria or inhibits cell multiplication. A reasonable explanation is that bacterial cells firmly attach to wool fibers forming a biofilm during multiplication. When the specimen was lifted off the nutrient agar surface, the cells in the form of biofilm remained attached to the wool fibers, removing the biomass and resulting in a clear, no growth zone underneath it. By imaging the textile specimens with X-ray microtomography, we concluded that the degree of attachment could be dependent on surface topography. The results indicate that certain textiles, in this case, wool, could exhibit antibacterial properties by removing excess bacteria that grow on the textile/skin interface when taken off the body.     

Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces

A newly discovered class of cell resistant surfaces, specifically engineered polyelectrolyte multilayers, was patterned with varying densities of adhesion ligands to control attachment of mammalian cells and to study the effects of ligand density on cell activity. Cell adhesive patterns were created on cell resistant multilayer films composed of poly(acrylic acid) and polyacrylamide through polymer-on-polymer stamping of poly(allylamine hydrochloride) PAH and subsequent reaction of the amine functional groups with an adhesion ligand containing RGD (Arg-Gly-Asp). These cell patterns demonstrated great promise for long-term applications since they remained stable for over 1 month, unlike ethylene glycol functional surfaces. By changing the stamping conditions of PAH, it was possible to alter the number of available functional groups in the patterned regions, and as a result, control the ligand density. Cell spreading, morphology, and cytoskeletal organization were compared at four different RGD densities. The highest RGD density, approximately 152 000 molecules/microm2, was created by stamping PAH at a pH of 11.0. Lowering the stamping ink pH led to patterns with lower ligand surface densities (83 000 molecules/microm2 for pH 9.0, 53,000 molecules/ microm2 for pH 7.0, and 25 000 molecules/microm2 for pH 3.5). An increasing number of cells attached and spread as the RGD density of the patterns increased. In addition, more cells showed well-defined actin stress fibers and focal adhesions at higher levels of RGD density. Finally, we found that pattern geometry affected cytoskeletal protein organization. Well-formed focal adhesions and cell-spanning stress fibers were only found in cells on wider line patterns (at least 25 microm in width).     

An interfacial oxime reaction to immobilize ligands and cells in patterns and gradients to photoactive surfaces

We report a molecularly controlled interfacial chemoselective methodology to immobilize ligands and cells in patterns and gradients to self-assembled monolayers on gold. This strategy is based on reacting soluble ketone or aldehyde tethered ligands to surface-bound oxyamine alkeanethiols to generate a covalent oxime linkage to the surface. We characterize the kinetic behavior of the reaction on the surface with ferrocenecarboxaldehyde (FcCHO) as a model ligand. The precise extent of immobilization and therefore surface density of FcCHO on the SAM is monitored and determined by cyclic voltammetry, which shows a peudo-first-order rate constant of 0.13 min(-1). In order to generate complex surface patterns and gradients of ligands on the surface, we photoprotected the oxyamine group with nitroveratryloxycarbonyl (NVOC). We show that ultraviolet light irradiation through a patterned microfiche film reveals the oxyamine group and we characterize the rate of deprotection by immobilization of ketone containing redox active groups. Finally, we extend this strategy to show biospecific cell attachment of fibroblast cells by immobilizing ketone-GRGDS peptides in patterns. The interfacial oxime reaction is chemoselective and stable at physiological conditions (pH 7.0, 37 degrees C) and may potentially be used to install ligands on the surface in the presence of attached cells to modulate the cell microenvironment to generate dynamic surfaces for monitoring changes in cell behavior in real time.     

Surface-enhanced Raman spectroscopy based on conical holed enhancing substrates

In this contribution, surface-enhanced Raman spectroscopy (SERS) based on conical holed glass substrates deposited with silver colloids was reported for the first time. It combines the advantages of both dry SERS assays based on plane films deposited with silver colloids and wet SERS assays utilizing cuvettes or capillary tubes. Compared with plane glass substrates deposited with silver colloids, the conical holed glass substrates deposited with silver colloids exhibited five-to ten-folds of increase in the rate of signal enhancement, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte of interest. The conical holed glass substrates in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy (MEM_SERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4% and a limit of detection of about 0.02 μM using a portable i-Raman 785H spectrometer. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEM_SERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc.     

Rails and anchors: guiding and trapping droplet microreactors in two dimensions

This paper presents a method to control the motion of nanolitre drops in a wide and thin microchannel, by etching fine patterns into the channel's top surface. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. The resulting gain in surface energy pulls a drop into the groove such that localized holes can be used as anchors for holding drops, while linear patterns can be used as rails to guide them along complex trajectories. An anchored drop can remain stationary indefinitely, as long as the driving flow rate is below a critical value which depends on the hole and drop sizes. By micro-fabricating holes into a grid pattern, drops can be arrayed and held in the observation field of a microscope against the mean carrier flow. Their contents can then be modulated by gas exchange with the flowing carrier oil. We demonstrate in particular how the pH or the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Oxygen control is used to selectively deoxygenate droplets that encapsulate red blood cells from patients suffering from sickle cell disease, in order to study the polymerization of intracellular hemoglobin. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological flows.     

Deciphering the impact of PEG antifouling layer on surface attached functional peptides in regulating cell behaviors

In the ‘noise’ environment of culture medium, a medium length PEG antifouling layer (the OEG8 layer) can effectively resist the non-specific cell adhesion and present the optimal and genuine function of peptides in controlling cell behaviors.     

Deciphering the impact of PEG antifouling layer on surface attached functional peptides in regulating cell behaviors

It is highly desired to have bioactive surfaces for biomaterials and controllable interactions with cells.These functions were widely achieved by attaching functional peptides to the surface of biomaterials.It is well known that an antifouling layer can help reducing the nonspecific cell attachment.However,it is unclear how an antifouling PEG layer affects the function of peptides attached on material surface in controlling cell behavior.This highlight introduced the recent JACS paper from Prof.Liu and coworkers in addressing this question thoroughly.