New Poly(N-isopropylacrylamide-butylacrylate) Copolymer Biointerfaces and Their Characteristic Influence on Cell Behavior In Vitro.

Designing and obtaining new synthetic smart biointerfaces with specific and controlled characteristics relevant for applications in biomedical and bioengineering domains represents one of the main challenges in these fields. In this work, Matrix-Assisted Pulsed Laser Evaporation (MAPLE) is used to obtain synthetic biointerfaces of poly(N-isopropyl acrylamide-butyl acrylate) p(NIPAM-BA) copolymer with different characteristics (i.e., roughness, porosity, wettability), and their effect on normal HEK 293 T and murine melanoma B16-F1 cells is studied. For this, the influence of various solvents (chloroform, dimethylsulfoxide, water) and fluence variation (250-450 mJ/cm2) on the morphological, roughness, wettability, and physico-chemical characteristics of the coatings are evaluated by atomic force microscopy, scanning electron microscopy, contact angle measurements, Fourier-transform-IR spectroscopy, and X-ray photoelectron spectroscopy. Coatings obtained by the spin coating method are used for reference. No significant alteration in the chemistry of the surfaces is observed for the coatings obtained by both methods. All p(NIPAM-BA) coatings show hydrophilic character, with the exception of those obtained with chloroform at 250 mJ/cm2. The surface morphology is shown to depend on both solvent type and laser fluence and it ranges from smooth surfaces to rough and porous ones. Physico-chemical and biological analysis reveal that the MAPLE deposition method with fluences of 350-450 mJ/cm2 when using DMSO solvent is more appropriate for bioengineering applications due to the surface characteristics (i.e., pore presence) and to the good compatibility with normal cells and cytotoxicity against melanoma cells.

Flow injection enzymatic biosensor for aldehydes based on a Meldola Blue-Ni complex electrochemical mediator.

Carbon nanofibers (CNF) are efficient electrode modifiers in electrochemical biosensors that enhance the electrochemical active area, induce electrocatalytic effect toward the oxidation of the enzymatic cofactor nicotinamide adenine dinucleotide (reduced form, NADH), and enable the quantitative immobilization of enzymes. Combining CNF with efficient and stable mediators radically augments the speed of electron transfer between NADH and solid electrodes and leads to electrochemical sensors characterized by high sensitivity and stability. The main aim of this work was to investigate the performance of a novel mediator for NADH with advantageously low solubility in an electrochemical detector based on a screen-printed CNF electrode as well as its potential in biosensing. Using a mediator, prepared from Meldola Blue and Ni hexamine chloride, a stable and sensitive electrochemical NADH sensor is provided with a detection limit of 0.5 µmol L-1. Further on, covalent immobilization of a recently described aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 strain on the surface of the mediator-modified electrode produced a stable biosensor for the detection of aldehydes. When integrated in a flow injection analysis (FIA) setup with amperometric detection at 0.1 V vs. Ag/AgCl, the measurement of benzaldehyde with a detection limit of 10 µmol L-1 over a linear range of 30-300 µmol L-1 is possible. Determination of trace benzaldehyde impurities in a pharmaceutical excipient was also demonstrated and results compared with a chromatographic method. Graphical abstract.

Induced Hydrophilicity and In Vitro Preliminary Osteoblast Response of Polyvinylidene Fluoride (PVDF) Coatings Obtained via MAPLE Deposition and Subsequent Thermal Treatment.

Recent advancements in biomedicine have focused on designing novel and stable interfaces that can drive a specific cellular response toward the requirements of medical devices or implants. Among these, in recent years, electroactive polymers (i.e., polyvinylidene fluoride or PVDF) have caught the attention within the biomedical applications sector, due to their insolubility, stability in biological media, in vitro and in vivo non-toxicity, or even piezoelectric properties. However, the main disadvantage of PVDF-based bio-interfaces is related to the absence of the functional groups on the fluoropolymer and their hydrophobic character leading to a deficiency of cell adhesion and proliferation. This work was aimed at obtaining hydrophilic functional PVDF polymer coatings by using, for the first time, the one-step, matrix-assisted pulsed evaporation (MAPLE) method, testing the need of a post-deposition thermal treatment and analyzing their preliminary capacity to support MC3T3-E1 pre-osteoblast cell survival. As osteoblast cells are known to prefer rough surfaces, MAPLE deposition parameters were studied for obtaining coatings with roughness of tens to hundreds of nm, while maintaining the chemical properties similar to those of the pristine material. The in vitro studies indicated that all surfaces supported the survival of viable osteoblasts with active metabolisms, similar to the "control" sample, with no major differences regarding the thermally treated materials; this eliminates the need to use a secondary step for obtaining hydrophilic PVDF coatings. The physical-chemical characteristics of the thin films, along with the in vitro analyses, suggest that MAPLE is an adequate technique for fabricating PVDF thin films for further bio-applications.

Love Wave Surface Acoustic Wave Sensor with Laser-Deposited Nanoporous Gold Sensitive Layer.

Laser-deposited gold immobilization layers with different porosities were incorporated into Love Wave Surface Acoustic Wave sensors (LW-SAWs). Acetylcholinesterase (AChE) enzyme was immobilized onto three gold interfaces with different morphologies, and the sensor response to chloroform was measured. The response of the sensors to various chloroform concentrations indicates that their sensing properties (sensitivity, limit of detection) are considerably improved when the gold layers are porous, in comparison to a conventional dense gold layer. The results obtained can be used to improve properties of SAW-based biosensors by controlling the nanostructure of the gold immobilization layer, in combination with other enzymes and proteins, since the design of the present sensor is the same as that for a Love Wave biosensor.

MAPLE Assembled Acetylcholinesterase⁻Polyethylenimine Hybrid and Multilayered Interfaces for Toxic Gases Detection.

Developing a controlled method for obtaining hybrid enzymatic-based interfaces for sensing application require the use of a multiuse, reusable sensor. By controlling the interface characteristics in terms of the surface chemistry, thickness, and roughness, a tailored response toward various toxic compounds can be obtained, regarding both materials used as active surfaces and fabrication methods. Herein, we report a preliminary study on using a laser-based method (i.e., matrix-assisted pulsed laser evaporation, or MAPLE) for obtaining active polymeric⁻enzymatic interfaces as hybrid or layered coatings for detecting toxic vapors. The MAPLE fabrication consisted of the simultaneous alternating evaporation of layers of polyethylenimine (PEI) and acetylcholinesterase (AchE) in order to obtain active surfaces as both hybrid PEI-AchE and a PEI/AchE layered coating, respectively. The deposition processes of the polymer and enzyme were carried out using a double-target system and a Nd:YAG pulsed laser, operating at 0.45 J/cm² fluences with a wavelength of 266 nm and a repetition rate of 10 Hz. Fourier transform infrared spectroscopy revealed no significant changes in the functional groups of both hybrid and layered coatings compared with the initial material. The thickness and roughness, as well as the morphologies of the coatings revealed by atomic force microscopy and scanning electron microscopy showed coatings thicker than two µm that had smooth surfaces and average roughness values below six nm. The sensors were tested with simulants for nerve gases and pesticides containing phosphonate ester groups, namely dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP), and a different sensitivity was shown to the selected chemical agents for each of the sensors. The best sensitivities for DMMP and DIMP obtained by using a PEI-AchE coated sensor are 65 kHz and 200 kHz, respectively, whereas the best sensitivity when using multilayered interfaces is 30 kHz and 10 KHz for DIMP and DMMP, respectively.

Development of a DsRed-expressing HepaRG cell line for real-time monitoring of hepatocyte-like cell differentiation by fluorescence imaging, with application in screening of novel geometric microstructured cell growth substrates.

The bipotent nature of the HepaRG cell line is a unique property among human hepatoma-derived cells. Cell treatment with specific differentiation inducers results in a mixture of hepatocyte- and biliary-like cells, accompanied by upregulation of liver-specific proteins, drug metabolizing enzymes, transcription regulators, membrane receptors or innate immune response effectors. These features make the HepaRG cells a suitable and handy replacement for primary hepatocytes, to study hepatic functions in vitro. However, cell differentiation is a long, variable process, requiring special culture conditions, while the resulting mixed cell populations is usually a major drawback. This process can potentially be controlled by interface characteristics, such as substrate topography. To screen for such novel substrates, we have first developed a new HepaRG cell line, designated as HepaRGDsRed, expressing the reporter gene DsRed. The fluorescent protein was expressed in hepatocyte- and not biliary-like cells, in a differentiation dependent-manner. We have further used replicated microstructured gradients of polydimethylsiloxane (PDMS) that allow three-dimensional manipulation in vitro, to monitor HepaRGDsRed differentiation in real time. We demonstrate that this approach enables the controlled assembly of viable hepatocyte-like cells for functional studies, which can be maintained in culture without loss of differentiation. The regulated expression of the DsRed reporter proved a valuable tool not only for rapid screening of novel cell growth substrates favoring cell differentiation, but also, to enrich the hepatocyte-like cell population by fluorescence-activated cell sorting to investigate liver-specific processes in vitro.

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.

Hybrid bio-nanoporous peptide loaded-polymer platforms with anticancer and antibacterial activities.

In this study, hybrid bio-nanoporous peptides loaded onto poly(N-isopropylacrylamide-co-butylacrylate) (pNIPAM-co-BA) coatings were designed and obtained via matrix-assisted pulsed laser evaporation (MAPLE) technique. The incorporation of cationic peptides magainin (MG) and melittin (Mel) and their combination was tailored to target synergistic anticancer and antibacterial activities with low toxicity on normal mammalian cells. Atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy as well as contact angle and surface energy measurements revealed the successful and functional incorporation of both the peptides within porous polymeric nanolayers as well as surface modifications (i.e. variation in the pore size diameter, surface roughness, and wettability) after Mel, MG or Mel-MG incorporation compared to pNIPAM-co-BA. In vitro testing revealed the impairment of biofilm formation on all the hybrid coatings while testing with S. aureus, E. coli and P. aeruginosa. Moreover, MG was shown to modulate the effect of Mel in the combined Mel-MG extract formulation released via pNIPAM-platforms, thus significantly reducing cancer cell proliferation through apoptosis/necrosis as revealed by flow cytometry analysis performed in vitro on HEK293T, A375, B16F1 and B16F10 cells. To the best of our knowledge, Mel-MG combination entrapped in the pNIPAM-co-BA copolymer has not yet been reported as a new promising candidate with anticancer and antibacterial properties for improved utility in the biomedical field. Mel-MG incorporation compared to pNIPAM-co-BA in in vitro testing revealed the impairment of biofilm formation in all the hybrid formulations.

Surface Modification Using MAPLE Technique for Improving the Mechanical Performance of Adhesive Joints.

The adhesive bonds that ensure the appropriate mechanical properties for metal joining imply the surface chemical and wetting modification characteristics of the substrates. In this work, matrix-assisted pulsed laser evaporation (MAPLE) was used for the surface modification of Al via the deposition of two chemical compounds, polyvinyl alcohol (PVA) and triethanolamine (TEA), from frozen aqueous solutions. The deposition of the TEA and PVA layers was evidenced by FT-IR, SEM, and AFM analysis. The contact angle measurements evidenced the change in the hydrophilicity of the surface and surface free energies. The performance of the commercial silyl-based polymer adhesive Bison Max Repair Extreme Adhesive® was evaluated by tensile strength measurements. This method led to a change in tensile strength of 54.22% in the case of Al-TEA and 36.34% for Al-PVA compared with the control. This study gives preliminary insights into using MAPLE, for the first time in adhesive applications, as a pretreatment method for Al plates for adhesive bonding reinforcement.

Chemistry-Induced Effects on Cell Behavior upon Plasma Treatment of pNIPAAM.

In the field of bioengineering, depending on the required application, the attachment of various biological entities to the biomaterial is either favored or needs to be prevented. Therefore, different surfaces modification strategies were developed in combination with the characteristics of the materials. The present contribution reports on the use of the specific surface property of a thermoresponsive polymer poly(N-isopropylacrylamide) pNIPAAM obtained by spin coating in combination with plasma treatment for tuning cell behavior on treated polymeric surfaces. Topographical information for the plasma-treated pNIPAAM coatings obtained by Atomic Force Microscopy (AFM) measurements evidenced a more compact surface for Ar treatment due to combined etching and redeposition, while for oxygen, a clear increase of pores diameter is noticed. The chemical surface composition as determined by X-ray Photoelectron Spectroscopy showed the specific modifications induced by plasma treatment, namely strong oxidation for oxygen plasma treatment illustrated by eight times increase of O-C=O contribution and respectively an increase of C-N/O=C-N bonds in the case of ammonia plasma treatment. Structural information provided by FTIR spectroscopy reveals a significant increase of the carboxylic group upon argon and mostly oxygen plasma treatment and the increase in width and intensity of the amide-related groups for the ammonia plasma treatment. The biological investigations evidenced that L929 fibroblast cells viability is increased by 25% upon plasma treatment, while the cell attachment is up to 2.8 times higher for the oxygen plasma-treated surface compared to the initial spin-coated pNIPAAM. Moreover, the cell detachment process proved to be up to 2-3 times faster for the oxygen and argon plasma-treated surfaces and up to 1.5 times faster for the ammonia-treated surface. These results show the versatility of plasma treatment for inducing beneficial chemical modifications of pNIPAAM surfaces that allows the tuning of cellular response for improving the attachment-detachment process in view of tissue engineering.

Mitigation of Cellular and Bacterial Adhesion on Laser Modified Poly (2-Methacryloyloxyethyl Phosphorylcholine)/Polydimethylsiloxane Surface.

Nowadays, using polymers with specific characteristics to coat the surface of a device to prevent undesired biological responses can represent an optimal strategy for developing new and more efficient implants for biomedical applications. Among them, zwitterionic phosphorylcholine-based polymers are of interest due to their properties to resist cell and bacterial adhesion. In this work, the Matrix-Assisted Laser Evaporation (MAPLE) technique was investigated as a new approach for functionalising Polydimethylsiloxane (PDMS) surfaces with zwitterionic poly(2-Methacryloyloxyethyl-Phosphorylcholine) (pMPC) polymer. Evaluation of the physical-chemical properties of the new coatings revealed that the technique proposed has the advantage of achieving uniform and homogeneous stable moderate hydrophilic pMPC thin layers onto hydrophobic PDMS without any pre-treatment, therefore avoiding the major disadvantage of hydrophobicity recovery. The capacity of modified PDMS surfaces to reduce bacterial adhesion and biofilm formation was tested for Gram-positive bacteria, Staphylococcus aureus (S. aureus), and Gram-negative bacteria, Escherichia coli (E. coli). Cell adhesion, proliferation and morphology of human THP-1 differentiated macrophages and human normal CCD-1070Sk fibroblasts on the different surfaces were also assessed. Biological in vitro investigation revealed a significantly reduced adherence on PDMS-pMPC of both E. coli (from 29 × 10 6 to 3 × 102 CFU/mL) and S. aureus (from 29 × 106 to 3 × 102 CFU/mL) bacterial strains. Additionally, coated surfaces induced a significant inhibition of biofilm formation, an effect observed mainly for E. coli. Moreover, the pMPC coatings improved the capacity of PDMS to reduce the adhesion and proliferation of human macrophages by 50% and of human fibroblast by 40% compared to unmodified scaffold, circumventing undesired cell responses such as inflammation and fibrosis. All these highlighted the potential for the new PDMS-pMPC interfaces obtained by MAPLE to be used in the biomedical field to design new PDMS-based implants exhibiting long-term hydrophilic profile stability and better mitigating foreign body response and microbial infection.

In Vitro Effect of Replicated Porous Polymeric Nano-MicroStructured Biointerfaces Characteristics on Macrophages Behavior.

In the last decades, optimizing implant properties in terms of materials and biointerface characteristics represents one of the main quests in biomedical research. Modifying and engineering polyvinylidene fluoride (PVDF) as scaffolds becomes more and more attractive to multiples areas of bio-applications (e.g., bone or cochlear implants). Nevertheless, the acceptance of an implant is affected by its inflammatory potency caused by surface-induced modification. Therefore, in this work, three types of nano-micro squared wells like PVDF structures (i.e., reversed pyramidal shape with depths from 0.8 to 2.5 microns) were obtained by replication, and the influence of their characteristics on the inflammatory response of human macrophages was investigated in vitro. FTIR and X-ray photoelectron spectroscopy analysis confirmed the maintaining chemical structures of the replicated surfaces, while the topographical surface characteristics were evaluated by AFM and SEM analysis. Contact angle and surface energy analysis indicated a modification from superhydrophobicity of casted materials to moderate hydrophobicity based on the structure's depth change. The effects induced by PVDF casted and micron-sized reversed pyramidal replicas on macrophages behavior were evaluated in normal and inflammatory conditions (lipopolysaccharide treatment) using colorimetric, microscopy, and ELISA methods. Our results demonstrate that the depth of the microstructured surface affects the activity of macrophages and that the modification of topography could influence both the hydrophobicity of the surface and the inflammatory response.

Thin Films of Metal-Organic Framework Interfaces Obtained by Laser Evaporation.

Properties such as large surface area, high pore volume, high chemical and thermal stability, and structural flexibility render zeolitic imidazolate frameworks (ZIFs) well-suited materials for gas separation, chemical sensors, and optical and electrical devices. For such applications, film processing is a prerequisite. Herein, matrix-assisted pulsed laser evaporation (MAPLE) was successfully used as a single-step deposition process to fabricate ZIF-8 films. By correlating laser fluency and controlling the specific transfer of lab-synthesized ZIF-8, films with user-controlled physical and chemical properties were obtained. Films' characteristics were evaluated by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The analysis showed that frameworks of ZIF-8 can be deposited successfully and controllably to yield polycrystalline films. The deposited films maintained the integrity of the individual ZIF-8 framework, while undergoing minor crystalline and surface chemistry changes. No significant changes in particle size were observed. Our study demonstrated control over both the MAPLE deposition conditions and the outcome, as well as the suitability of the listed deposition method to create composite architectures that could potentially be used in applications ranging from selective membranes to gas sensors.

Lactoferrin in Bone Tissue Regeneration.

Among the multiple properties exhibited by lactoferrin (Lf), its involvement in bone regeneration processes is of great interest at the present time. A series of in vitro and in vivo studies have revealed the ability of Lf to promote survival, proliferation and differentiation of osteoblast cells and to inhibit bone resorption mediated by osteoclasts. Although the mechanism underlying the action of Lf in bone cells is still not fully elucidated, it has been shown that its mode of action leading to the survival of osteoblasts is complemented by its mitogenic effect. Activation of several signalling pathways and gene expression, in an LRPdependent or independent manner, has been identified. Unlike the effects on osteoblasts, the action on osteoclasts is different, with Lf leading to a total arrest of osteoclastogenesis. Due to the positive effect of Lf on osteoblasts, the potential use of Lf alone or in combination with different biologically active compounds in bone tissue regeneration and the treatment of bone diseases is of great interest. Since the bioavailability of Lf in vivo is poor, a nanotechnology- based strategy to improve the biological properties of Lf was developed. The investigated formulations include incorporation of Lf into collagen membranes, gelatin hydrogel, liposomes, loading onto nanofibers, porous microspheres, or coating onto silica/titan based implants. Lf has also been coupled with other biologically active compounds such as biomimetic hydroxyapatite, in order to improve the efficacy of biomaterials used in the regulation of bone homeostasis. This review aims to provide an up-to-date review of research on the involvement of Lf in bone growth and healing and on its use as a potential therapeutic factor in bone tissue regeneration.

Bioinstructive Micro-Nanotextured Zirconia Ceramic Interfaces for Guiding and Stimulating an Osteogenic Response In Vitro.

Osseous implantology's material requirements include a lack of potential for inducing allergic disorders and providing both functional and esthetic features for the patient's benefit. Despite being bioinert, Zirconia ceramics have become a candidate of interest to be used as an alternative to titanium dental and cochlear bone-anchored hearing aid (BAHA) implants, implying the need for endowing the surface with biologically instructive properties by changing basic parameters such as surface texture. Within this context, we propose anisotropic and isotropic patterns (linear microgroove arrays, and superimposed crossline microgroove arrays, respectively) textured in zirconia substrates, as bioinstructive interfaces to guide the cytoskeletal organization of human mesenchymal stem cells (hMSCs). The designed textured micro-nano interfaces with either steep ridges and microgratings or curved edges, and nanoroughened walls obtained by direct femtosecond laser texturing are used to evaluate the hMSC response parameters and osteogenic differentiation to each topography. Our results show parallel micro line anisotropic surfaces are able to guide cell growth only for the steep surfaces, while the curved ones reduce the initial response and show the lowest osteogenic response. An improved osteogenic phenotype of hMSCs is obtained when grown onto isotropic grid/pillar-like patterns, showing an improved cell coverage and Ca/P ratio, with direct implications for BAHA prosthetic development, or other future applications in regenerating bone defects.

Human Mesenchymal Stem Cell Response to Lactoferrin-based Composite Coatings.

The potential of mesenchymal stem cells (MSCs) for implantology and cell-based therapy represents one of the major ongoing research subjects within the last decades. In bone regeneration applications, the various environmental factors including bioactive compounds such as growth factors, chemicals and physical characteristics of biointerfaces are the key factors in controlling and regulating osteogenic differentiation from MSCs. In our study, we have investigated the influence of Lactoferrin (Lf) and Hydroxyapatite (HA) embedded within a biodegradable PEG-PCL copolymer on the osteogenic fate of MSCs, previous studies revealing an anti-inflammatory potential of the coating and osteogenic differentiation of murine pre-osteoblast cells. The copolymer matrix was obtained by the Matrix Assisted Pulsed Laser Evaporation technique (MAPLE) and the composite layers containing the bioactive compounds (Lf, HA, and Lf-HA) were characterised by Scanning Electron Microscopy and Atomic Force Microscopy. Energy-dispersive X-ray spectroscopy contact angle and surface energy of the analysed coatings were also measured. The characteristics of the composite surfaces were correlated with the viability, proliferation, and morphology of human MSCs (hMSCs) cultured on the developed coatings. All surfaces were found not to exhibit toxicity, as confirmed by the LIVE/DEAD assay. The Lf-HA composite exhibited an increase in osteogenic differentiation of hMSCs, results supported by alkaline phosphatase and mineralisation assays. This is the first report of the capacity of biodegradable composite layers containing Lf to induce osteogenic differentiation from hMSCs, a property revealing its potential for application in bone regeneration.

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 lysozyme's 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.

Graphene nanoplatelets-sericin surface-modified Gum alloy for improved biological response.

In this study a "Gum Metal" titanium-based alloy, Ti-31.7Nb-6.21Zr-1.4Fe-0.16O, was synthesized by melting and characterized in order to evaluate its potential for biomedical applications. The results showed that the newly developed alloy presents a very high strength, high plasticity and a low Young's modulus relative to titanium alloys currently used in medicine. For further bone implant applications, the newly synthesized alloy was surface modified with graphene nanoplatelets (GNP), sericin (SS) and graphene nanoplatelets/sericine (GNP-SS) composite films via Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. The characterization of each specimen was monitored by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA) measurements, and Fourier Transform Infrared Spectroscopy (FTIR). The materials' surface analyses suggested the successful coating of GNP, SS and GNP-SS onto the alloy surface. Additionally, the activities of pre-osteoblasts such as cell adhesion, cytoskeleton organization, cell proliferation and differentiation potentials exhibited on these substrates were investigated. Results showed that the GNP-SS-coated substrate significantly enhanced the growth and osteogenic differentiation of MC3T3-E1 cells when compared to bare and GNP-coated alloy. Collectively, the results show that GNP-SS surface-modified Gum alloy can modulate the bioactivity of the pre-osteoblasts holding promise for improved biological response in vivo.

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.