V. Dinca

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Microfabrication of polystyrene microbead arrays by laser induced forward transfer

In this study we describe a simple method to fabricate microarrays of polystyrene microbeads (PS-μbeads) on Thermanox coverslip surfaces using laser induced forward transfer (LIFT). A triazene polymer layer which acts as a dynamic release layer and propels the closely packed microspheres on the receiving substrate was used for this approach. The deposited features were characterized by optical microscopy, scanning electron microscopy, atomic force microscopy, and Raman spectroscopy. Ultrasonication was used to test the adherence of the transferred beads. In addition, the laser ejection of the PS-μbead pixels was investigated by time resolved shadowgraphy. It was found that stable PS-μbeads micropatterns without any specific immobilization process could be realized by LIFT. These results highlight the increasing role of LIFT in the development of biomaterials, drug delivery, and tissue engineering.

MAPLE-based method to obtain biodegradable hybrid polymeric thin films with embedded antitumoral agents

In this work, antitumor compounds, lactoferrin [recombinant iron-free (Apo-rLf)], cisplatin (Cis) or their combination were embedded within a biodegradable polycaprolactone (PCL) polymer thin film, by a modified approach of a laser-based technique, matrix-assisted pulsed laser evaporation (MAPLE). The structural and morphological properties of the deposited hybrid films were analyzed by Fourier-transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). The in vitro effect on the cells’ morphology and proliferation of murine melanoma B16-F10 cells was investigated and correlated with the films’ surface chemistry and topography. Biological assays revealed decreased viability and proliferation, lower adherence, and morphological modifications in the case of melanoma cells cultured on both Apo-rLf and Cis thin films. The antitumor effect was enhanced by deposition of Apo-rLf with Cis within the same film. The unique capability of the new approach, based on MAPLE, to embed antitumor active factors within a biodegradable matrix for obtaining novel biodegradable hybrid platform with increased antitumor efficiency has been demonstrated.

Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces

Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples.

Applications of laser printing for organic electronics

The development of organic electronic requires a non contact digital printing process. The European funded e-LIFT project investigated the possibility of using the Laser Induced Forward Transfer (LIFT) technique to address this field of applications. This process has been optimized for the deposition of functional organic and inorganic materials in liquid and solid phase, and a set of polymer dynamic release layer (DRL) has been developed to allow a safe transfer of a large range of thin films. Then, some specific applications related to the development of heterogeneous integration in organic electronics have been addressed. We demonstrated the ability of LIFT process to print thin film of organic semiconductor and to realize Organic Thin Film Transistors (OTFT) with mobilities as high as 4 10-2 cm2.V-1.s-1 and Ion/Ioff ratio of 2.8 105. Polymer Light Emitting Diodes (PLED) have been laser printed by transferring in a single step process a stack of thin films, leading to the fabrication of red, blue green PLEDs with luminance ranging from 145 cd.m-2 to 540 cd.m-2. Then, chemical sensors and biosensors have been fabricated by printing polymers and proteins on Surface Acoustic Wave (SAW) devices. The ability of LIFT to transfer several sensing elements on a same device with high resolution allows improving the selectivity of these sensors and biosensors. Gas sensors based on the deposition of semiconducting oxide (SnO2) and biosensors for the detection of herbicides relying on the printing of proteins have also been realized and their performances overcome those of commercial devices. At last, we successfully laser-printed thermoelectric materials and realized microgenerators for energy harvesting applications.

2D and 3D biotin patterning by ultrafast lasers

Bio-micro-array fabrication and biological molecules patterning has been the focus of much research in recent years, as they are envisaged to play an important part in genomic studies, drug discovery and screening, protein identification and scaffolding development for tissue engineering. A number of different approaches have been examined for fabricating patterned biological surfaces. Almost in all cases, patterning of biomolecules has been two-dimensional. We demonstrate both 2D and 3D biotin patterning using techniques which enable the construction of arbitrary two and three dimensional shapes, not restricted to array-based shapes. For the 2D printing, Laser Induced Forward Transfer (LIFT) is employed to deposit controlled and viable micro-patterns of biotin. The activity and the functionality of the transferred materials are shown. For the 3D printing, firstly micro-structures are made employing multi-photon polymerisation. Biotin is subsequently immobilised on the structures surface by excimer laser photo-activation of photobiotin and further incubated with fluorescent labelled streptavidin. The specificity of the binding is demonstrated. The methods allow not only prototyping but also direct device construction.

Construction of 2D and 3D biomolecules structures using fs lasers

Bio-microarray fabrication has been the focus of much research in recent years, as they are envisaged to play an important part in genomic studies, protein identification, drug discovery and screening. We demonstrate both 2D and 3D biomolecules patterning using techniques which enable the construction of arbitrary two and three-dimensional shapes, not restricted to array-based shapes. For the 2D printing, Laser-Induced Forward Transfer (LIFT) is employed to deposit controlled and viable micropatterns of the Horseradish Peroxidase (HRP) and the functionality of the transferred materials is shown. For the 3D printing, firstly microstructures are made employing multiphoton polymerisation and then biotin is subsequently immobilised on the surface of the structures by excimer laser photo-activation of photobiotin and further exposed to fluorescently labelled streptavidin. The specificity of the binding is demonstrated.

2D and 3D printing of biomolecules employing femtosecond lasers

We demonstrate two- and three-dimensional patterning of biological molecules. For the two-dimensional patterning we employ Laser-Induced Forward Transfer of materials in solution. For the three-dimensional patterning we employ femtosecond-laser induced three-photon polymerization, a technique which enables the construction of arbitrary 2D and 3D structures of submicron resolution. Biotin is subsequently attached to the 3D structures via UV-activated crosslinking. The integrity of the photolytically immobilized biotin is confirmed by detecting the binding of fluorescently labeled avidin via fluorescence microscopy.

Functional Micrococcus lysodeikticus layers deposited by laser technique for the optical sensing of lysozyme

Whole cell optical biosensors, made by immobilizing whole algal, bacterial or mammalian cells on various supports have found applications in several fields, from ecology and ecotoxicity testing to biopharmaceutical production or medical diagnostics. We hereby report the deposition of functional bacterial layers of Micrococcus lysodeikticus (ML) via Matrix-Assisted Pulsed Laser Evaporation (MAPLE) on poly(diallyldimethylamonium) (PDDA)-coated-glass slides and their application as an optical biosensor for the detection of lysozyme in serum. Lysozyme is an enzyme upregulated in inflammatory diseases and ML is an enzymatic substrate for this enzyme. The MAPLE-deposited bacterial interfaces were characterised by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Fourier-Transformed Infrared Spectroscopy (FTIR), Raman and optical microscopy and were compared with control interfaces deposited via layer-by-layer on the same substrate. After MAPLE deposition and coating with graphene oxide (GO), ML-modified interfaces retained their functionality and sensitivity to lysozymes lytic action. The optical biosensor detected lysozyme in undiluted serum in the clinically relevant range up to 10μgmL-1, in a fast and simple manner.