Biosurface engineering through ink jet printing

The feasibility of thermal ink jet printing as a robust process for biosurface engineering was demonstrated. The strategy investigated was to reconstruct a commercial printer and take advantage of its colour management interface. High printing resolution was achieved by formulating bio-inks of viscosity and surface tension similar to those of commercial inks. Protein and enzyme denaturation during thermal ink jet printing was shown to be insignificant. This is because the time spent by the biomolecules in the heating zone of the printer is negligible; in addition, the air and substrate of high heat capacity absorb any residual heat from the droplet.Gradients of trophic/tropic factors can serve as driving force for cell growth or migration for tissue regeneration. Concentration gradients of proteins were printed on scaffolds to show the capability of ink jet printing. The printed proteins did not desorb upon prolonged immersion in aqueous solutions, thus allowing printed scaffold to be used under in vitro and in vivo conditions. Our group portrait was ink jet printed with a protein on paper, illustrating that complex biopatterns can be printed on large area. Finally, patterns of enzymes were ink jet printed within the detection and reaction zones of a paper diagnostic.     

Estrogen receptor microarrays: subtype-selective ligand binding

We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.     

Micro-Scale Cell Patterning on Nonfouling Plasma Polymerized Tetraglyme Coatings by Protein Microcontact Printing

Nonfouling thin films were prepared by the plasma deposition of tetraethylene glycol dimethyl ether (pp4G) on fluorinated ethylene propylene polymer (FEP) and glass substrates. Ordered cell patterns were created on these surfaces by microcontact printing of proteins. Pp4G was found to be stable in aqueous environments and resistant to an ethanol sterilization procedure, as verified by surface analysis. Pp4G also reduced nonspecific protein adsorption by more than 65-fold before and after sterilization. Despite the low adsorption of proteins to pp4G in solution, protein microcontact printing was achieved and we were able to print laminin, an adhesive extracellular matrix protein, from an elastomeric stamp onto pp4G. The printed laminin supported the attachment and spreading of cardiomyocytes and the nonprinted pp4G regions remained cell repulsive in culture conditions. Microscale patterns of cardiomyocytes were maintained on printed pp4G for more than 7 days. This cell patterning process should be viable for other cell types. The potential applications include tissue engineering and microdevices for biosensor, diagnostic, and pharmacological applications.     

Printing biomacromolecules on a bovine serum albumin precursor layer

Various biomacromolecules including proteins and polysaccharides are printed on a substrate capped with a bovine serum albumin (BSA) precursor layer to create clear co-patterns of these molecules. Characterizations by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) demonstrate the successful production and clear boundaries of the co-patterns. Rinsing the BSA-adsorbed substrate and the biomacromolecules-inked stamp before microcontact printing (microCP) is crucial for the creation of clear and stable co-patterns. The patterns are mainly stabilized by electrostatic interactions and van der Waals forces. Characterizations by ellipsometry, UV-Vis and fluorescence spectroscopy reveal that printing by a flat PDMS stamp yields a denser layered structure of proteins with a higher amount than that of adsorbed proteins. By printing, however, a lower enzymatic catalytic activity for horseradish peroxidase (HRP) or binding capability for avidin (both normalized to amount) is determined. A conformational transition from alpha-helix to beta-sheet of HRP is observed by ATR-IR. By contrast, a BSA precursor layer can effectively improve the functionality of the printed HRP or avidin and preserve the original conformation of the proteins, although the absolute transferred amount of these proteins is decreased.     

Imaging of affinity microcontact printed proteins by using liquid crystals

This paper reports the design of surfaces on which thermotropic liquid crystals can be used to image affinity microcontact printed proteins. The surfaces comprise gold films deposited onto silica substrates at an oblique angle of incidence and then functionalized with a monolayer formed from 2-mercaptoethylamine. Ellipsometric measurements confirm the transfer of anti-biotin IgG to these surfaces from affinity stamps functionalized with biotinylated bovine serum albumin (BSA), while control experiments performed using anti-goat IgG confirmed the specificity of the IgG capture on the stamp. On these surfaces, anti-biotin IgG caused nematic phases of 4-cyano-4'-pentylbiphenyl (5CB, Delta epsilon = epsilon(parallel) - epsilon(perpendicular) > 0) to assume orientations that were parallel to the surfaces (planar anchoring) but with azimuthal orientations that were distinct from those assumed by the liquid crystals on the amine-terminated surfaces not supporting IgGs. Following incubation of these samples for >8 h at 36 degrees C, we observed that the amine-terminated regions of the surface not supporting IgG cause 5CB to undergo a transition from planar to perpendicular (homeotropic). Because N-(4-methoxybenzylidene)-4-butylaniline (MBBA) (Delta epsilon < 0) does not undergo a similar transition in orientation, this transition is consistent with the effects of an electrical double layer formed at the amine-terminated surface on the liquid crystal. Following the transition to homeotropic anchoring, the liquid crystals provide high optical contrast between regions of the surface supporting and not supporting IgG. We conclude that amine-terminated surfaces (I) uniformly align liquid crystals when not supporting proteins and (II) have sufficiently high surface free energy to capture proteins delivered to the surface from an affinity stamp, and thus they form the basis of a useful class of surfaces on which affinity microcontact printed proteins can be imaged using liquid crystals.     

Circumventing the problems caused by protein diversity in microarrays: implications for protein interaction networks

Protein microarrays provide a well-controlled, high-throughput way to uncover protein-protein interactions. One problem with this and other standardized assays, however, is that proteins vary considerably with respect to their physical properties. If a simple threshold-based approach is used to define protein-protein interactions, the resulting binary networks can be strongly biased. Here, we investigate the extent to which even closely related protein interaction domains vary when printed as microarrays. We find that, when a collection of well behaved, monomeric Src homology 2 (SH2) domains are printed at the same concentration, they vary by up to 50-fold with respect to the resulting surface density of active protein. When a threshold-based binding assay is performed on these domains using fluorescently labeled phosphopeptides, a misleading picture of the underlying biophysical interactions emerges. This problem can be circumvented, however, by obtaining saturation binding curves for each protein-peptide interaction. Importantly, the equilibrium dissociation constants obtained from these curves are independent of the surface density of active protein. We submit that an increased emphasis should be placed on obtaining quantitative information from protein microarrays and that this should serve as a more general goal in all efforts to define large-scale protein interaction networks.     

Fabrication of antibody arrays using thermally responsive elastin fusion proteins

A general method has been developed to immobilize antibodies onto an array surface by employing fusion proteins consisting of an elastin domain with tunable hydrophobic properties and an antibody-binding domain with high binding affinity and specificity for antibodies. Antibodies conjugated with the elastin fusion proteins can be directly printed on a self-assembled monolayer-modified glass slide in a functionally active orientation with a spatially defined pattern. An antibody array sensor for detection of tumor markers was fabricated to demonstrate the utility of the method. We expect that the method presented here could be a simple and universal platform to immobilize antibodies for the fabrication of a variety of antibody array sensors.     

Electroanalytical sensing of chromium(III) and (VI) utilising gold screen printed macro electrodes

We report the fabrication of gold screen printed macro electrodes which are electrochemically characterised and contrasted to polycrystalline gold macroelectrodes with their potential analytical application towards the sensing of chromium(III) and (VI) critically explored. It is found that while these gold screen printed macro electrodes have electrode kinetics typically one order of magnitude lower than polycrystalline gold macroelectrodes as is measured via a standard redox probe, in terms of analytical sensing, these gold screen printed macro electrodes mimic polycrystalline gold in terms of their analytical performance towards the sensing of chromium(III) and (VI), whilst boasting additional advantages over the macro electrode due to their disposable one-shot nature and the ease of mass production. An additional advantage of these gold screen printed macro electrodes compared to polycrystalline gold is the alleviation of the requirement to potential cycle the latter to form the required gold oxide which aids in the simplification of the analytical protocol. We demonstrate that gold screen printed macro electrodes allow the low micro-molar sensing of chromium(VI) in aqueous solutions over the range 10 to 1600 μM with a limit of detection (3σ) of 4.4 μM. The feasibility of the analytical protocol is also tested through chromium(VI) detection in environmental samples.     

Disposable highly ordered pyrolytic graphite-like electrodes: Tailoring the electrochemical reactivity of screen printed electrodes

We demonstrate that the electron transfer properties of disposable screen printed electrodes can be readily tailored via the introduction of a polymeric formulation into the ink used to fabricate these electrochemical platforms. This approach allows the role of the binder on the underpinning electrochemical properties to be explored and allows the electrochemical reactivity of the screen printed electrodes to be tailored from that of edge plane to basal plane of highly ordered pyrolytic graphite.     

Fabricating protein immunoassay arrays on nitrocellulose using dip-pen lithography techniques

Advancements in lithography methods for printing biomolecules on surfaces are proving to be potentially beneficial for disease screening and biological research. Dip-pen nanolithography (DPN) is a versatile micro and nanofabrication technique that has the ability to produce functional biomolecule arrays. The greatest advantage, with respect to the printing mechanism, is that DPN adheres to the sensitive mild conditions required for biomolecules such as proteins. We have developed an optimised, high-throughput printing technique for fabricating protein arrays using DPN. This study highlights the fabrication of a prostate specific antigen (PSA) immunoassay detectable by fluorescence. Spot sizes are typically no larger than 8 μm in diameter and limits of detection for PSA are comparable with a commercially available ELISA kit. Furthermore, atomic force microscopy (AFM) analysis of the array surface gives great insight into how the nitrocellulose substrate functions to retain protein integrity. This is the first report of protein arrays being printed on nitrocellulose using the DPN technique and the smallest feature size yet to be achieved on this type of surface. This method offers a significant advance in the ability to produce dense protein arrays on nitrocellulose which are suitable for disease screening using standard fluorescence detection.     

Fabrication of substrates with defined mechanical properties and topographical features for the study of cell migration

Both substrate topography and substrate mechanical properties are known to influence cell behavior, but little is known about how they act in concert. Here, a method is presented to introduce topographical features into PA hydrogel substrates that span a wide range of physiological E values. Gel swelling plays a significant role in the fidelity of protruding micromolded features, with the most efficient pattern transfer occurring at a crosslinking concentration equal to or greater than ≈5%. In contrast, swelling does not influence the spacing fidelity of microcontact printed islands of collagen on 2D PA substrates. BAECs cultured on micromolded PA substrates exhibit contact guidance along ridges patterned for all E tested.     

Covalent microcontact printing of proteins for cell patterning

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An aldehyde-terminated monolayer on glass or on gold was obtained by the reaction between an amino-terminated monolayer and terephthaldialdehyde. The aldehyde monolayer was employed as a substrate for the direct microcontact printing of bioengineered, collagen-like proteins by using an oxidized poly(dimethylsiloxane) (PDMS) stamp. After immobilization of the proteins into adhesive "islands", the remaining areas were blocked with amino-poly(ethylene glycol), which forms a layer that is resistant to cell adhesion. Human malignant carcinoma (HeLa) cells were seeded and incubated onto the patterned substrate. It was found that these cells adhere to and spread selectively on the protein islands, and avoid the poly(ethylene glycol) (PEG) zones. These findings illustrate the importance of microcontact printing as a method for positioning proteins at surfaces and demonstrate the scope of controlled surface chemistry to direct cell adhesion.     

Characterization and fabrication of disposable screen printed microelectrodes

We report the fabrication of disposable and flexible screen printed microelectrodes which are characterised with microscopy and cyclic voltammetry. These new type of screen printed electrochemical platforms consist of micro-sized graphite typically with radii of 60 to 100 microns are defined by an inert dielectric. The advantage of this type of electrochemical sensing platform is that each microelectrode is disposable and cost effective and thus does not require extensive cleaning or electrode pre-treatment between measurements. Prior to measurements the screen printed microelectrode needs only to be calibrated with a suitable redox probe, as is typically the case with microelectrodes. We show proof of concept that the screen printed microelectrodes are advantageous for electro-analytical measurements with the example of determination of lead via cathodic stripping voltammetry. The use of graphite screen printed microelectrodes allows comparable detection limits to that obtained in the literature at insonated boron doped diamond electrodes, without the need for power ultrasound – which otherwise limits the widespread applicability and ease of measurement.     

Smooth and flexible filler-nanocellulose composite structure for printed electronics applications

A new type of micro/nanocomposite was made by using only micro fibrillated cellulose and inorganic fillers. This composite structure can contain up to 90% fillers being still mechanically stable and flexible. Calendering can be used to produce dense structures with extremely smooth surface. To study the effect of filler shape and type, both kaolin and precipitated calcium carbonate (PCC) based sheets were examined. Microscopy (cross-sectional and surface SEM images) and mechanical and morphological properties, including strength properties, surface roughness and dimensional stability as a function of moisture were analysed. After calendering the surface of the PCC containing sheets was smoother than that of photopaper and in the same level as reference plastic film Mylar A. The dimensional stability of the sheets was clearly better than that of paper sheets. The combination of a good dimensional stability with low surface roughness makes these structures potential for printed electronics applications, in which they could replace oil-based plastic substrates. Suitability for printed electronic applications was tested by inkjet printing conductors with silver nanoparticle ink. The sheet resistances of conductors printed on kaolin based sheets were close to those printed on plastic Mylar A film.     

Reversible Hydrophobic Barriers Introduced by Microcontact Printing: Application to Protein Microarrays

Microcontact printing (µCP) has been used to introduce temporary hydrophobic barriers on carboxymethylated dextran (CMD) hydrogels on gold. Among the investigated types of inks, tetraoctadecylammonium bromide (TOAB), electrostatically bound to the CMD layer, provided the most well-defined features both with respect to pattern-definition and reversibility upon exposure to a regeneration solution. The printed patterns were characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM), microscopic wetting and imaging null ellipsometry to explore the influence of concentration of ink solution and contact time on the appearance of the printed layer. AFM revealed that the printed TOAB molecules aggregated into clusters rather than into a homogeneous mono- or multilayer on the CMD hydrogel. It was also observed that printed areas of TOAB that are larger than 25µm are inhomogeneous most likely because of an edge transfer lithography (ETL) mechanism. A protein model system based on Protein A-rabbit antimouse Fc was used to evaluate the potential of the patterned surface as a protein microarray chip by means of surface plasmon microscopy (SPM). Moreover, non-specific adsorption of several proteins onto TOAB barriers was also studied using surface plasmon resonance (SPR), and it is evident that undesired adsorption can be eliminated by removing barriers after ligand immobilization, but prior to analyte exposure, by treating the patterned surface with a simple salt regeneration solution.     

Base-atom recognition in protein adsorption to alkyl agaroses.

The three proteins phosphorylase b, calmodulin and fibrinogen are adsorbed onto thioalkyl derivatives of Sepharose much more strongly than onto gels carrying the same alkyl residue coupled via a carbamate linkage. This enhancement of binding onto alkyl-S-Sepharoses compared with alkyl-N-agaroses is not primarily due to an increase in the extent of conformational changes of the proteins occurring on the gel surface. This can be shown in experiments with the tripeptide Trp-Trp-Trp. The Trp tripeptide is also adsorbed with a much higher affinity to butyl-S-Sepharose than to butyl-N-Sepharose, showing that the primary interaction between the immobilized alkyl residue and the amino acids of the protein is decisive for adsorption. A model stressing the strong influence of an atom or a group of atoms at the base of an immobilized alkyl residue is described as "base-atom recognition".     

A robust small-molecule microarray platform for screening cell lysates

Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.     

A simple, disposable microfluidic device for rapid protein concentration and purification via direct-printing

A facile and disposable microfluidic device for rapid protein concentration was fabricated by using a direct printing process. Two printed V-shaped microchannels in mirror image orientation were separated by a 100 mum wide toner gap. When a high electric field was applied across the two channels, nanofissures were formed by electric breakdown at the junction toner gap. This microfluidic device with nanofissures was used as a concentrator for protein. Negatively charged proteins were observed to concentrate at the anode side of the nanofissures upon application of an electric field across this junction. Using this device, about 10(3)-10(5)-fold protein concentration was achieved within 10 min. Systematic investigation showed that the concentration mechanism could be explained by the ion exclusion-enrichment effect of the nanofissures. In addition, the present microchip device integrated both functions of concentration and purification were confirmed. This simple on chip protein preconcentration and purification device could be a disposable sample preparation component in printed microfluidic systems used for practical biochemical assays.     

Influence of N-methylation on a cation-pi interaction produces a remarkably stable beta-hairpin peptide

The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.     

A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo

We discovered a class of naturally occurring human proteins with unusually high net positive charge that can potently deliver proteins in functional form into mammalian cells both in?vitro and also in murine retina, pancreas, and white adipose tissues in?vivo. These findings represent diverse macromolecule delivery agents for in?vivo applications, and also raise the possibility that some of these human proteins may penetrate cells as part of their native biological functions.