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Nanofluidic systems offer a huge potential for discovery of new molecular transport and chemical phenomena that can be employed for future technologies. Herein, we report on the transport behavior of surface-reactive compounds in a nanometer-scale flow of phospholipids from a scanning probe. We have investigated microscopic deposit formation on polycrystalline gold by lithographic printing and writing of 1,2-dioleoyl-sn-glycero-3-phosphocholine and eicosanethiol mixtures, with the latter compound being a model case for self-assembled monolayers (SAMs). By analyzing the ink transport rates, we found that the transfer of thiols was fully controlled by the fluid lipid matrix allowing to achieve a certain jetting regime, i.e., transport rates previously not reported in dip-pen nanolithography (DPN) studies on surface-reactive, SAM-forming molecules. Such a transport behavior deviated significantly from the so-called molecular diffusion models, and it was most obvious at the high writing speeds, close to 100 mu m s(-1). Moreover, the combined data from imaging ellipsometry, scanning electron microscopy, atomic force microscopy (AFM), and spectroscopy revealed a rapid and efficient ink phase separation occurring in the AFM tip-gold contact zone. The force curve analysis indicated formation of a mixed ink meniscus behaving as a self-organizing liquid. Based on our data, it has to be considered as one of the co-acting mechanisms driving the surface reactions and self-assembly under such highly nonequilibrium, crowded environment conditions. The results of the present study significantly extend the capabilities of DPN using standard AFM instrumentation: in the writing regime, the patterning speed was already comparable to that achievable by using electron beam systems. We demonstrate that lipid flow-controlled chemical patterning process is directly applicable for rapid prototyping of solid-state devices having mesoscopic features as well as for biomolecular architectures.

Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.

Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single- or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

An efficient procedure for chemical initiator-free, in situ synthesis of a functional polyethylene glycol methacrylate (PEG MA) hydrogel on regular glass substrates is reported. It is demonstrated that self-initiated photografting and photopolymerization driven by UV irradiation can yield tens of nanometer-thick coatings of carboxy-functionalized PEG MA on the aldehyde-terminated borosilicate glass surface. The most efficient formulation for hydrogel synthesis contained methyl methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), and PEG methacrylate (PEG10MA) monomers (1:1:1). The resulting HEMA/PEG10MA/MAA (HPMAA) coatings had a defined thickness in the range from 11 to 50 nm. The physicochemical properties of the synthesized HPMAA coatings were analyzed by combining water contact angle measurements, stylus profilometry, imaging null ellipsometry, and atomic force microscopy (AFM). The latter technique was employed in the quantitative imaging mode not only for direct probing of the surface topography but also for swelling behavior characterization in the pH range from 4.5 to 8.0. The estimated high swelling ratios of the HPMAA hydrogel (up to 3.2) together with its good stability and resistance to nonspecific protein binding were advantageous in extracellular matrix mimetics via patterning of fibronectin (FN) at a resolution close to 200 nm. It was shown that the fabricated FN micropatterns on HPMAA were equally suitable for single-cell arraying, as well as controlled cell culture lasting at least for 96 h.

We developed an experimentally simple method for localized N-tert-butyloxycarbonyl amino group (N-Boc) deprotection on surface applicable to meso-scale on-demand patterning for further synthetic modifications. The chemical patterning is accomplished on heated self-assembled monolayers using micro-drop equipment and water as the sole chemical.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Electropolymerisation of riboflavin (Rf) was performed in different media in order to optimise the best conditions for the creation of a stable and electroactive polymerised riboflavin (PRf) film. Electropolymerisation was performed using cyclic voltammetry (CV) in solutions with various pH in different potential windows. Different electrode substrates were used for PRf deposition: pyrolytic graphite (PG) and highly oriented pyrolytic graphite (HOPG). The obtained films were characterised spectroscopically, microscopically and electrochemically applying Raman spectroscopy, reflection-absorption infrared spectroscopy (RAIRS), atomic force microscopy (AFM) and electrochemical methods such as cyclic voltammetry (CV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) in different media. The best film was obtained when PRf was electropolymerised from 0.1 mol L −1 PBS, pH 7.0. Raman and RAIRS data have indicated involvement of isoalloxazine ring III in the polymerisation process. The best response of such an electrode was in the same buffer solution. This electrode will be further applied in (bio)sensor construction.

Cobalt-manganese ferrites were synthesized by the mechanochemical synthesis using metal salts as a precursor and sodium chloride as a growth agent. The dependence of the size and the magnetic properties of the prepared nanoparticles on the high-energy ball milling conditions were investigated. Cobalt-manganese ferrite nanoparticles were characterized using the Mossbauer spectroscopy, atomic force microscopy (AFM) and X-ray diffraction. Measurements showed that the particle size of the cobalt ferrite nanoparticles varied from 4 nm up to ∼10 nm without and with the addition of sodium chloride respectively whereas no such effect of the sodium chloride on manganese nanoparticles was observed.

The femtosecond pulses offer a flexible way to induce internal modifications inside fused silica and prepare embedded 3D microchannel structures. In this paper, an internal to external microfluidic device is presented. The micro-channels were prepared with the direct laser writing technique using different scanning methods. The sample was immersed in diluted 10 % concentration HF acid to remove the modified material. A few independent 3D microchannels were inscribed, and each of them was connected to a pump to circulate the protein solution during the measurement preserving a constant flow rate. Microchannels were designed to act as flow cells in ellipsometric imaging surface plasmon resonance (iSPR) setup to study the real-time interaction kinetics of biological materials (proteins or DNA). The microchannels allow to make few parallel measurements at the same time and minimize the required biological sample amounts for the iSPR experiment.

Folic acid was polymerised electrochemically at a glassy carbon electrode surface from 0.1 mol L−1 phosphate buffer saline solution, pH 5.0, containing 0.1 mmol L−1 monomer. The obtained thin film was porous with a pore size of 50–60 nm. Since its electrochemical stability was rather short, the polyfolate film was covered with a graphene-chitosan composite layer which increased its stability significantly. The best strategy to immobilise the enzyme was crosslinking with glutaraldehyde. The lifetime of this glucose biosensor in use was at least 12 days, on-shelf life time was at least 30 days. The linear range was up to 1 mmol L−1 and the LOD was 0.6 µmol L−1. The first polyfolate-based biosensor was applied to analysis of natural samples.

Although three-dimensional shaping of metallic nanostructures is an important strategy for control and manipulation of localized surface plasmon resonance (LSPR), its implementation in high-throughput, on-chip fabrication of plasmonic devices remains challenging. Here, we demonstrate nanocontact-based large-area fabrication of a novel, LSPR-active Au architecture consisting of periodic arrays of reduced-symmetry nanoantennas having sub-50 nm, out-of-plane features. Namely, by combining nanosphere and molecular self-assembly processes, we have patterned evaporated polycrystalline Au films for chemical etching of nanocups with controlled aspect ratios (outer diameter d = 100 nm and void volumes = 18 or 39 zL). The resulting nanoantennas were highly ordered, forming a hexagonal lattice structure over centimeter-sized glass substrates, and they displayed characteristic LSPR absorption in the visible/near-infrared spectral range. Theoretical simulations indicated electric field confinement and enhancement patterns located not only around the rims but also inside the nanocups. We also explored how these patterns and the overall spectral characteristics depended on the nanocup aspect ratio as well as on electric field coupling in the arrays. We have successfully tested the fabricated architecture for detection of stepwise immobilization and interactions of proteins, thus demonstrating its potential for both nanoscopic scaffolding and sensing of biomolecular assemblies.

We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures.

The results of patterning of the indium–tin oxide (ITO) film on the glass substrate with high repetition rate picosecond lasers at various wavelengths are presented. Laser radiation initiated the ablation of the material, forming grooves in ITO. Profile of the grooves was analyzed with a phase contrast optical microscope, a stylus type profiler, scanning electron microscope (SEM) and atomic force microscope (AFM). Clean removal of the ITO film was achieved with the 266 nm radiation when laser fluence was above the threshold at 0.20 J/cm2, while for the 355 nm radiation, the threshold was higher, above 0.46 J/cm2. The glass substrate was damaged in the area where the fluence was higher than 1.55 J/cm2. The 532 nm radiation allowed getting well defined grooves, but a lot of residues in the form of dust were generated on the surface. UV radiation with the 266 nm wavelength provided the widest working window for ITO ablation without damage of the substrate. Use of UV laser radiation with fluences close to the ablation threshold made it possible to minimize surface contamination and the recast ridge formation during the process.

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 of 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 500 μm thick RHCIII-MPC constructs comprising over 85% water, are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, as compared to the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30 μm wide fibronectin stripes enhanced cell attachment and showed 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. We thank Dr. Chyan-Jang Lee for establishing the GFP-HCEC cell line used for this study, and Ms. Kimberley Merrett for assistance in characterization of the hydrogels. We also thank Dr. Sadhana Kulkani and David Priest, University of Ottawa Eye Institue, for assistance with the laser cutting study; and Dr. Joanne M. Hackett (currently at Cambridge University Health Partners) for assistance with preliminary cell culture/biocompatibility studies during optimization of the RHCIII-MPC hydrogels. We thank Johannes Junger and Michael Baumann, MLase AG, for help with the UV crosslinking, and the Medical Devices Bureau, Health Canada, for use of the SEM system. We gratefully acknowledge funding from an NSERC-CIHR Canada Collaborative Health Research Project grant (M.G.) and subsequent funding for an EU Nanomedicine ERAnet project "I-CARE" to M.G., R.V. and MLase AG, through the Swedish Research Council, Research Council of Lithuania and VDI Germany, respectively.

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.

All laser components can withstand a limited intensity of optical radiation and the measurement of laser-induced damage thresholds (LIDT) is required. In the case of repetitive pulses the LIDT measurements should be performed according ISO 11254-2 standard. This procedure is time consuming and puts a high requirement on human resources. In order to speed up the LIDT measurements with a minimal human resource we developed the automated station for LIDT measurements according ISO-11254-2 standard. In this paper we overview the main parts of this automated station and present the results of LIDT measurements with repetitive pulses. In order to control the LIDT measurements, software based on LabView programming package was created. The LIDT software controls experimental sample positioning in X and Y directions, laser pulse energy attenuation and shutter. It also automates damage detection and performs statistical analysis. The program recognizes damage by detecting scattered light from damaged surface. The input of sample and laser beam technical parameters is required to start the measurements. The minimal distance between test sites on the sample surface is calculated automatically, and the surface area is divided in to a hexagonal matrix. The program also chooses the laser pulse train energy for each test site. The program also allows fast damage inspection by translating the sample under the Nomarski microscope. After completion of measurement and damage inspection, the program automatically generates the measurement report.

In this paper, we performed comparison of laser-induced damage threshold (LIDT) for ion-assisted deposition (IAD) and non-IAD coatings in nanosecond and femtosecond pulse ranges. S-on-I tests were performed for 10/sup 4/ pulses at 1-kHz repetition rate. Equivalent BK7 substrates with the same surface roughness were coated for comparison of the two coating. The results were analysed using AFM and XRD. The difference in LIDT values for 130 fs pulses between the IAD optical coating and non-IAD coating deposited on the substrate with the same polishing quality was equal within range of measurement accuracy. In contrast to femtosecond pulses the LIDT values for the nanosecond pulses showed stronger and opposite correlation with the deposition methods.

High density, improved adhesion and environmental stability are the main features of dielectric optical coatings produced using ion-assisted deposition (IAD) technology. However, investigations of resistance of IAD coatings to intensive laser radiation show controversial results. A series of experiments were done to examine the influence of ion gun operation on the transmittance of fused silica substrates. It was shown that operation of ion source introduced extinction in UV spectral range. Optical properties of single hafnia layers and multilayer dielectric mirrors deposited using conventional e-beam evaporation and different modes of IAD were investigated. Microstructural analysis using X-ray diffraction (XRD) measurements and AFM scanning of coated areas was carried out. Single hafnia layers deposited using high energy ion assistance had more amorphous structure with smaller crystallites of monoclinic phase. High reflection UV mirrors deposited using high energy ion assistance had slightly higher mean refractive indices of hafnia, higher extinction than conventional e-beam deposition, but demonstrated slightly higher laser induced damage threshold (LIDT) values measured at 355 nm. Deposition using the lowest energy ions produced the most porous coatings with the best LIDT of 7.7 J/cm 2 .

The ion assisted thin film deposition (IAD) method has been used extensively for more than two decades, but questions about possibility of improving of the laser-induced damage threshold (LIDT) by this method compared with the conventional electron-beam evaporation (non-IAD) method are still not fully answered. A more complete understanding of different factors that can influence laser-induced damage threshold is necessary for continued development of multilayer dielectric coatings optimized for high-power laser applications. To clarify these factors we performed comparison of LIDT for IAD and non-IAD coatings in nanosecond and femtosecond pulse ranges. High reflectance mirrors at 800 nm and 532 nm were tested. Mirror coatings were made of ZrO 2 and SiO 2 . Automated LIDT measurements were performed according to the requirements of current ISO 11254-2 standard. Two lasers were used for the measurements: Nd:YAG (λ = 532 nm, τ = 5 ns) and Ti:Sapphire (λ = 800 nm, τ = 130 fs). Measurements at 800 nm and 532 nm were performed at 1-kHz and 10 Hz pulse repetition rate respectively (S-on-1 test). The damage morphology of coatings was characterized by Nomarski microscopy and relation of LIDT with coating parameters was analyzed.

A comparison of laser induced damage thresholds (LIDT) of ion assisted deposition (IAD) and standard electron beam deposition dielectric coatings on BK7 glass with different surface roughness was performed. Five types of high reflectance mirrors at 800 nm and two types of high reflectance mirrors at 1064 nm were tested. Mirror coatings were made of ZrO 2 and SiO 2 . Automated LIDT measurements were performed according to the requirements of current ISO 11254-2 standard. Two lasers were used for the measurements: Nd:YAG (l = 1064 nm, t = 13 ns) and Ti:Sapphire (l = 800 nm, t = 130 fs ). All measurements were performed at 1-kHz pulse repetition rate (S-on-1 test). A fixed spot size was used for each laser. For 1064 nm it was ~ 70 um and for 800 nm ~ 500 um. The damage morphology and structure of coatings were characterized by an atomic force microscopy (AFM), Nomarski microscopy and X-ray diffraction (XRD).

For the development of standard measurement procedures in optics characterization, comparative measurement campaigns (Round-robin experiments) are indispensable. Within the framework of the CHOCLAB project in the mid-90s, several international Round-robins were successfully performed qualifying procedures for e. g. 1 on 1-LIDT, laser-calorimetry and total scattering. During the recent years, the demand for single pulse damage investigations has been overtaken by the more practically relevant S on 1-LIDT. In contrast to the industrial needs, the comparability of the multiple-pulse LIDT has not been proven by Round-robin experiments up to now. As a consequence of the current research activities on the interaction of ultra-short pulses with matter as well as industrial applications, numerous fs-laser systems become available in universities and research institutes. Furthermore, special problems for damage testing may be expected because of the intrinsic effects connected with the interaction of ultrashort pulses with optical materials. Therefore, a Round-robin experiment on S on 1-damage testing utilizing fs-pulses was conducted within the framework of the EUREKA-project CHOCLAB II. For this experiment, seven parties investigated different types of mirrors and windows. Most of the partners were guided by the International Standard ISO 11254-2, but one partner employed his own damage testing technique. In this presentation, the results of this comparative experiment are compiled demonstrating the problems induced by special effects of damage testing in the ultra-short pulse regime.

We report on the development and use of coherent spectrophotometers specialized for the unusual requirements of characterizing nonlinear optical materials and multilayer dielectric coatings used in laser systems. A large dynamic range is required to measure the linear properties of transmission, reflection and absorption and nonlinear properties of laser-induced damage threshold and nonlinear frequency conversion. Optical parametric oscillators generate coherent radiation that is widely tunable with instantaneous powers that can range from milliwatts to megawatts and are well matched to this application. As particular example a laser spectrophotometer based on optical parametric oscillators and a diode-pumped, Q-switched Nd:YAG laser and suitable for optical characterization in the spectral range 420-4500 nm is described. Measurements include reflectance and transmittance, absorption, scattering and laser-induced damage thresholds. Possibilities of a system based on a 130-fs Ti:sapphire laser and optical parametric generators are also discussed.