Ramūnas Valiokas

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.

Functional fabrication of recombinant human collagen–phosphorylcholine hydrogels for regenerative medicine applications

The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500μm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30μm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.

Differential protein assembly on micropatterned surfaces with tailored molecular and surface multivalency.

Life is a thing taken for granted by most. However, it is the life-long quest of many to unravel the mysteries of it. Understanding and characterizing the incomprehensively complex molecular interaction networks within a biological organism, which defines that organism, is a vital prerequisite to understand life itself. Already, there has been a lot of research conducted and a large knowledge has been obtained about these pathways over, especially, the last century. We have seen the fruits of these labors in e.g. the development of medicines which have been able to cure or at least arrest many diseases and conditions. However, many diseases are still incurable (e.g. cancer) and a lot more work is still needed for understanding them fully and designing successful treatments. This work describes a generic analytical tool platform for aiding in more efficient (bio)molecular interaction mapping analyses; protein microarray chips. Microarray chips are surfaces with micrometer sized features with the possibility of studying the interactions of many (thousands to tens of thousands) (bio)molecules in parallel. This allows for a higher throughput of analyses to be performed at a reduced time and cost. Protein microarrays have been around for approximately a decade, following in the footsteps of the, so far, more successfully used DNA microarrays (developed in the 1990s). Microarrays of proteins are more difficult to produce because of the more complex nature of proteins as compared to DNA. In our work we have constructed model surfaces which allow for the stable, highly oriented, and functional immobilization of proteins in an array format. Our capture molecules are based on multivalent units of the chelator nitrilotriacetic acid (NTA), which is able to bind histidine-tagged proteins. Furthermore, we have explored an approach for studying lipid membrane bound systems, e.g. receptor-ligand interactions, in a parallelized, microarray format. The approach relies on the addressable, DNA-mediated adsorption of tagged lipid vesicles. In an analogous work we have used the protein microarray concept for the detection of four common narcotics (heroin, amphetamine, ecstasy, and cocaine). The detection is based on the displacement of loosely bound antibodies from surface array positions upon injection of a specific target analyte, i.e. a narcotic substance. The proof-of-concept chip can easily be expanded to monitor many more narcotic substances. In addition, we have also been able to simultaneously detect the explosive trinitrotoluene (TNT) along with the narcotics, showing that the chip is a versatile platform for the detection of virtually any type of harmful or illegal compound. This type of biosensor system is potentially envisaged to be used in the fight against crime, terrorism, drug abuse etc. Infrared reflection absorption spectroscopy together with ellipsometry has been used to characterize molecular layers used in the fabrication processes of the microarray features. Imaging surface plasmon resonance operating in the ellipsometric mode is subsequently used for functional evaluation of the microarrays using a well-defined receptor-ligand model system. This approach allows simultaneous and continuous monitoring of binding events taking place in multiple regions of interest on the microarray chip. A common characteristic of all the instrumentation used is that there is no requirement for labeling of the biomolecules to be detected, e.g. with fluorescent or radioactive probes. This feature allows for a flexible assay design and the use of more native proteins, without any time-consuming pretreatments.

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.

Ab initio calculations of equilibrium geometries and vibrational excitations of helical ethylene-glycol oligomers: application to modeling of monolayer infrared spectra

The density functional theory methods are used to calculate the equilibrium molecular structures and vibrational spectra of helical H(CH2CH2O)nH (OEG) oligomers (n = 4-7) at a level of precision th ...

Functional Hydrogel Density Patterns Fabricated by Dip‐Pen Nanolithography and Photografting

This licentiate thesis is focused on two methods for protein immobilization to biosensor surfaces for future applications in protein microarray formats. The common denominator is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM) of functionalized alkanethiolates. Both methods involve photochemistry, in the first case for direct immobilization of proteins to the surface, in the other for grafting a hydrogel, which is then used for protein immobilization.Paper I describes the development and characterization of Chelation Assisted Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent attachment and controlled orientation of the immobilized recognition molecule (ligand) and thereby provides a robust sensor surface for detection of analyte in solution. The concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance (SPR) analysis, the binding ability of this bivalent ligand was found to be more than two times higher with random orientation obtained by amine coupling than with homogeneous orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to orientation effects than a monovalent ligand and that island formation of the alkanethiolates used for CAP results in a locally high ligand density and steric hindrance.Paper II describes the development of nanoscale hydrogel structures. These were photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG) chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV light irradiation and acted as a sacrificial layer. In this process other OEG residues were exposed and acted as new starting points for the self-initiated photografting and photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel density pattern and interaction with streptavidin was demonstrated by epifluorescence microscopy.

Structural and kinetic properties of laterally stabilized, oligo(ethylene glycol)-containing alkylthiolates on gold: A modular approach

The formation of highly ordered self-assembled monolayers (SAMs) on gold from an unusually long and linear compound HS(CH2)15CONH(CH2CH2O)6CH2CONH(CH2)15CH3 is investigated by contact angle goniometry, ex situ null ellipsometry, cyclic voltammetry and infrared reflection-absorption spectroscopy. The molecules are found to assemble in an upright position as a complete monolayer within 60 min. The overall structure of the SAM reaches equilibrium within 24 h as evidenced by infrared spectroscopy, although a slight improvement in water contact angles is observed over a period of a few weeks. The resulting SAM is 60 Å thick and it displays an advancing water contact angle of 112° and excellent electrochemical blocking characteristics with typical current densities about 20 times lower as compared to those observed for HS(CH2)15CH3 SAMs. The dominating crystalline phases of the supporting HS(CH2)15 and terminal (CH2)15CH3 alkyl portions, as well as the sealed oligo(ethylene glycol) (OEG) “core,” appear as unusually sharp features in the infrared spectra at room temperature. For example, the splitting seen for the CH3 stretching and CH2 scissoring peaks is normally only observed for conformationally trapped alkylthiolate SAMs at low temperatures and for highly crystalline polymethylenes. Temperature-programmed infrared spectroscopy in ultrahigh vacuum reveals a significantly improved thermal stability of the SAM under investigation, as compared to two analogous OEG derivatives without the extended alkyl chain. Our study points out the advantages of adopting a “modular approach” in designing novel SAM-forming compounds with precisely positioned in plane stabilizing groups. We demonstrate also the potential of using the above set of compounds in the fabrication of “hydrogel-like” arrays with controlled wetting properties for application in the ever-growing fields of protein and cell analysis, as well as for bioanalytical applications.

Lipid dip-pen nanolithography on self-assembled monolayers

Dip-pen nanolithography (DPN) with lipids as an ink enables functional micro/nanopatterning on different substrates at high process speeds. However, only a few studies have addressed the influence of the physicochemical properties of the surface on the structure and phase behavior of DPN-printed lipid assemblies. Therefore, by combining the scanning probe and optical imaging techniques in this work we have analyzed lipid microdomain formation on the self-assembled monolayers (SAMs) on gold as well-defined model surfaces that displayed hydrophilic (protein-repellent) or hydrophobic (protein-adhesive) characteristics. We have found that on the tri(ethylene glycol)-terminated SAM the lipid ink transfer was fast (~10–1 μm3 s−1), quasi-linear and it yielded unstable, sparsely packed lipid microspots. Contrary to this, on the methyl-terminated SAM the lipid transfer was ~20 times slower, nonlinear, and the obtained stable dots of ~1 μm in diameter consisted of lipid multilayers. Our comparative analysis indicated that the measured lipid transfer was consistent with the previously reported so-called polymer transfer model (Felts et al 2012, Nanotechnology 23 215301). Further on, by employing the observed distinct contrast in the DPN ink behavior we constructed confined lipid microdomains on pre-patterned SAMs, in which the lipids assembled either into monolayer or multilamellar phases. Such microdomains can be further utilized for lipid membrane mimetics in microarray and lab-on-a-chip device formats.