Surface-Enhanced Raman Scattering Using 2D Materials.

The use of surface-enhanced Raman scattering (SERS) as a technique for detecting small amounts of (bio)chemical analytes has become increasingly popular in various fields. While gold and silver nanostructures have been extensively studied as SERS substrates, the availability of other types of substrates is currently expanding the applications of this spectroscopic method. Recently, researchers have begun exploring two-dimensional (2D) materials (e. g., graphene-like nanostructures) as substrates for SERS analysis. These materials offer unique optical properties, a well-defined structure, and the ability to modify their surface chemistry. As a contribution to advance this field, this concept article highlights the significance of understanding the chemical mechanism that underlies the experimental Raman spectra of chemisorbed molecules onto 2D materials' surfaces. Therefore, the article discusses recent advancements in fabricating substrates using 2D layered materials and the synergic effects of using their metallic composites for SERS applications. Additionally, it provides a new perspective on using Raman imaging in developing 2D materials as analytical platforms for Raman spectroscopy, an exciting emerging research area with significant potential.

Surface-enhanced Raman scattering detection of thiram and ciprofloxacin using chitosan-silver coated paper substrates.

Fast detection of contaminants of emerging concern (CECs) in water resources is of great environmental interest. Ideally, sustainable materials should be used in water quality monitoring technologies implemented for such purposes. In this regard, the application of bio-based materials aimed at the fabrication of analytical platforms has become of great importance. This research merges both endeavors by exploring the application of chitosan-coated paper, decorated with silver nanoparticles (AgNPs), on surface-enhanced Raman scattering (SERS) spectroscopy studies of two distinct types of CECs dissolved in aqueous samples: an antibiotic (ciprofloxacin) and a pesticide (thiram). Our results indicate the superior SERS performance of biocoated substrates compared to their non-coated paper counterparts. The detection limits achieved for thiram and ciprofloxacin using the biocoated substrates were 0.024 ppm and 7.7 ppm, respectively. The efficient detection of both analytes is interpreted in terms of the role of the biopolymer in promoting AgNPs assemblies that result in local regions of enhanced SERS activity. Taking advantage of these observations, we use confocal Raman microscopy to obtain Raman images of the substrates using ciprofloxacin and thiram as molecular probes. We also demonstrate that these biobased substrates can be promising for on-site analysis when used in conjunction with portable Raman instruments.

Insightful vibrational imaging study on the hydration mechanism of carbamazepine.

Anhydrous carbamazepine (CBZ) is an anti-convulsant drug commonly used to treat epilepsy and relieve trigeminal neuralgia. The presence of the dihydrate form in commercial CBZ tablets can change the dissolution rate of the active pharmaceutical ingredient (API), thus decreasing its activity. The hydration transformation can occur during wet granulation or storage, within a few weeks, depending on the ambient conditions. This work aims to investigate the effect of relative humidity (RH) in the transition of pure anhydrous CBZ (CBZ III) into the hydrate form by using confocal Raman microscopy with cluster analysis (CA). Firstly, several tablets of pure CBZ III containing different amounts of CBZ DH (50%, 10%, 1%, 0.5%) were prepared and analyzed by Raman imaging with CA. Our results show that CBZ DH crystals can be detected in the CBZ III tablets, at as low a concentration as 0.5%, giving distinct Raman features for the analysed polymorphs. The stability of pure anhydrous (CBZ III) tablets was then monitored by Raman imaging at room temperature (20-22 °C) and different RH (6%, 60% and 89%). The Raman imaging with CA showed that the anhydrous CBZ tablets start to convert into the hydrate form after 48 h, and it completely changes after 120 hours (5 days) at RH 89%. The tablets exposed to RH 6% and 60% did not demonstrate the presence of CBZ DH after 1 week of exposure. The exposure time was extended for 9 months in the former, and no CBZ DH was observed. A comparative study using IR imaging was also performed, demonstrating the viability of these vibrational imaging techniques as valuable tools to monitor the hydration process of active pharmaceutical ingredients.

Effects of Graphene Oxide and Reduced Graphene Oxide Nanostructures on CD4+ Th2 Lymphocytes.

The activation of T helper (Th) lymphocytes is necessary for the adaptive immune response as they contribute to the stimulation of B cells (for the secretion of antibodies) and macrophages (for phagocytosis and destruction of pathogens) and are necessary for cytotoxic T-cell activation to kill infected target cells. For these issues, Th lymphocytes must be converted into Th effector cells after their stimulation through their surface receptors TCR/CD3 (by binding to peptide-major histocompatibility complex localized on antigen-presenting cells) and the CD4 co-receptor. After stimulation, Th cells proliferate and differentiate into subpopulations, like Th1, Th2 or Th17, with different functions during the adaptative immune response. Due to the central role of the activation of Th lymphocytes for an accurate adaptative immune response and considering recent preclinical advances in the use of nanomaterials to enhance T-cell therapy, we evaluated in vitro the effects of graphene oxide (GO) and two types of reduced GO (rGO15 and rGO30) nanostructures on the Th2 lymphocyte cell line SR.D10. This cell line offers the possibility of studying their activation threshold by employing soluble antibodies against TCR/CD3 and against CD4, as well as the simultaneous activation of these two receptors. In the present study, the effects of GO, rGO15 and rGO30 on the activation/proliferation rate of these Th2 lymphocytes have been analyzed by studying cell viability, cell cycle phases, intracellular content of reactive oxygen species (ROS) and cytokine secretion. High lymphocyte viability values were obtained after treatment with these nanostructures, as well as increased proliferation in the presence of rGOs. Moreover, rGO15 treatment decreased the intracellular ROS content of Th2 cells in all stimulated conditions. The analysis of these parameters showed that the presence of these GO and rGO nanostructures did not alter the response of Th2 lymphocytes.

A comparative study on emergent pollutants photo-assisted degradation using ruthenium modified titanate nanotubes and nanowires as catalysts.

Several methods have been used to tailor nanomaterials structure and properties. Sometimes, slight changes in the structure outcomes expressive improvements in the optical and photocatalytic properties of semiconductor nanoparticles. In this context, the influence of the metal doping and the morphology on a catalyst performance was studied in this work. Here, ruthenium doped titanate nanotubes (RuTNT) were synthesised for the first time using an amorphous Ru-containing precursor. Afterwards, the photocatalytic performance of this sample was compared to the one obtained for ruthenium titanate nanowires (RuTNW), recently reported. Two samples, RuTNW and RuTNT, were produced using the same Ru-containing precursor but distinct hydrothermal methodologies. The powders were structural, morphological and optical characterized by X-ray diffraction and fluorescence, transmission electron microscopy, Raman, X-ray photoelectron and photoluminescence spectroscopies. Distinct variations on the structural and optical properties of the RuTNT and RuTNW nanoparticles, due to ruthenium incorporation were observed. Their potential use as photocatalysts was evaluated on the hydroxyl radical photo-assisted production. Both samples were catalytic for this reaction, presenting better performances than the pristine counterparts, being RuTNT the best photocatalyst. Subsequently, the degradation of two emergent pollutants, caffeine and sulfamethazine, was studied. RuTNT demonstrated to be better photocatalyst than RuTNW for caffeine but identical performances were obtained for sulfamethazine. For both catalysts, the degradation mechanism of the pollutants was explored through the identification and quantification of the intermediate compounds produced and several differences were found. This indicates the importance of the structural and morphological aspects of a material on its catalytic performance.

Hybrid nanostructures for SERS: materials development and chemical detection.

This review focuses on recent developments in hybrid and nanostructured substrates for SERS (surface-enhanced Raman scattering) studies. Thus substrates composed of at least two distinct types of materials, in which one is a SERS active metal, are considered here aiming at their use as platforms for chemical detection in a variety of contexts. Fundamental aspects related to the SERS effect and plasmonic behaviour of nanometals are briefly introduced. The materials described include polymer nanocomposites containing metal nanoparticles and coupled inorganic nanophases. Chemical approaches to tailor the morphological features of these substrates in order to get high SERS activity are reviewed. Finally, some perspectives for practical applications in the context of chemical detection of analytes using such hybrid platforms are presented.

Integrated magneto-plasmonic nanostructures-based immunoassay for galectin-3 detection.

Cardiovascular diseases remain a leading cause of global mortality, highlighting the need for accurate diagnostic tools and the detection of specific cardiac biomarkers. Surface-enhanced Raman scattering (SERS) spectroscopy has proved to be a promising alternative diagnostic tool to detect relevant biomarkers compared to traditional methods. To our knowledge, SERS methodology has never been used to detect galectin-3 (Gal-3), a crucial biomarker for cardiovascular conditions. Our study aimed to develop plasmonic and magneto-plasmonic nanoplatforms for the sensitive immunodetection of Gal-3 using SERS. Spherical gold nanoparticles (AuNPs) were synthesized and functionalized with 11-mercaptoundecanoic acid (MUDA) to enable antibody binding and 4-mercaptobenzoic acid (4MBA) that served as a Raman reporter due to its intense Raman signal. Following bioconjugation with Gal-3 antibody, the AuNPs were employed in the immunodetection of Gal-3 in phosphate-buffer saline (PBS) solution, offering a limit of detection (LOD) of 12.2 ng mL-1 and a working range up to 120 ng mL-1. Furthermore, our SERS-based immunosystem demonstrated selectivity for Gal-3 (40 ng mL-1) in the presence of other biomolecules such as α-amylase, bovine serum albumin and human C-reactive protein. As a proof of concept, we developed magneto-plasmonic nanoparticles composed of silica-coated magnetite decorated with the bioconjugated AuNPs aimed at enhancing the uptake and detection of Gal-3 via SERS coupled with Raman imaging. Our findings underscore the potential of SERS-based techniques for the sensitive and specific detection of biomarkers, holding significant implications for improved diagnosis and surveillance of cardiovascular diseases. Future research will focus on further optimizing these nanoplatforms and their translation into clinical settings.

Corrigendum: Colloidal nanomaterials for water quality improvement and monitoring.

[This corrects the article DOI: 10.3389/fchem.2022.1011186.].

Carbamazepine polymorphism: A re-visitation using Raman imaging.

Raman imaging methods have appeared in the last years as a powerful approach to monitoring the quality of pharmaceutical compounds. Because polymorphism occurs in many crystalline pharmaceutical compounds, it is essential to monitor polymorphic transformations induced by different external stimulus, such as temperature changes, to which those compounds may be submitted. Raman imaging with k-means cluster analysis (CA) is used here as an essential technique to investigate structural and chemical transformations occurring in carbamazepine p-monoclinic (CBZ III) into carbamazepine triclinic (CBZ I) when submitted to temperatures near the melting point of CBZ III (178 °C) and CBZ I (193 °C). CBZ III commercial powder and laboratorial prepared CBZ I were analyzed by differential scanning calorimetry, powder X-ray diffraction and Raman spectroscopy with variable temperature. After thermal treatment, the resultant CBZ powder was evaluated by Raman imaging, in which all imaging data was analyzed using CA. Raman imaging allowed the identification of different polymorphs of CBZ (CBZ III and CBZ I) and iminostilbene (IMS), a degradation product of CBZ, in the treated samples, depending on the heating treatment method.

Dendrimer stabilized nanoalloys for inkjet printing of surface-enhanced Raman scattering substrates.

Research on paper substrates prepared by inkjet deposition of metal nanoparticles for sensing applications has become a hot topic in recent years; however, the design of such substrates based on the deposition of alloy nanoparticles remains less explored. Herein, we report for the first time the inkjet printing of dendrimer-stabilized colloidal metal nanoalloys for the preparation of paper substrates for surface-enhanced Raman scattering (SERS) spectroscopy. To this end, nanoassemblies containing variable molar ratios of Au:Ag were prepared in the presence of poly(amidoamine) dendrimer (PAMAM), resulting in plasmonic properties that depend on the chemical composition of the final materials. The dendrimer-stabilized Au:Ag:PAMAM colloids exhibit high colloidal stability, making them suitable for the preparation of inks for long-term use in inkjet printing of paper substrates. Moreover, the pre-treatment of paper with a polystyrene (PS) aqueous emulsion resulted in hydrophobic substrates with improved SERS sensitivity, as illustrated in the analytical detection of tetramethylthiuram disulfide (thiram pesticide) dissolved in aqueous solutions. We suggest that the interactions established between the two polymers (PAMAM and PS) in an interface region over the cellulosic fibres, resulted in more exposed metallic surfaces for the adsorption of the analyte molecules. The resulting hydrophobic substrates show long-term plasmonic stability with high SERS signal retention for at least ninety days.

Modification of Paper Surface by All-Lignin Coating Formulations.

All-lignin coating formulations were prepared while combining water-soluble cationic kraft lignin (quaternized LignoBoost®, CL) and anionic lignosulphonate (LS). The electrostatic attraction between positively charged CL and negatively charged LS led to the formation of insoluble self-organized macromolecule aggregates that align to films. The structures of the formed layers were evaluated by atomic force microscopy (AFM), firstly on glass lamina using dip-coating deposition and then on handsheets and industrial uncoated paper using roll-to-roll coating in a layer-by-layer mode. Coated samples were also characterized by optical microscopy, scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (SEM/EDS), and contact angle measurements. It was suggested that the structure of all-lignin aggregates is the result of the interaction of amphiphilic water-soluble lignin molecules leading to their specifically ordered mutual arrangement depending on the order and the mode of their application on the surface. The all-lignin coating of cellulosic fiber imparts lower air permeability and lower free surface energy to paper, mainly due to a decrease in surface polarity, thus promoting the paper's hydrophobic properties. Moderate loading of lignin coating formulations (5-6 g m-2) did not affect the mechanical strength of the paper.

Interfacial assembly of zinc(II) phthalocyanines on graphene oxide (GO): Stable "turn-off-on" nanoplatforms to detect G-quadruplexes (G4).

HYPOTHESIS: The aggregation of phthalocyanines (Pcs) enfeebles their suitability as G-quadruplex (G4) ligands over time. It is hypothesized that the interfacial assembly of Pcs on graphene oxide (GO) influences intermolecular interactions, thereby affecting their physicochemical properties and inducing stabilization of Pcs in solution. Hence, the stacking of Pcs on GO could be tuned to create nanosystems with the ability to detect G4 for longer periods through a slow release of Pcs. EXPERIMENTS: Four cationic structurally-related zinc(II) phthalocyanines (ZnPc) were non-covalently assembled on GO by ultrasonic exfoliation. A comprehensive characterization of ZnPcs@GO was carried out by spectroscopic techniques and electron microscopy to understand the organization of ZnPcs on GO. The fluorescence of ZnPcs@GO was studied in the presence of G4 (T2G5T)4 and duplex ds26 through spectrofluorimetric titrations and monitored along time. FINDINGS: GO induced a re-organization of the ZnPcs mostly to J-aggregates and quenched their original fluorescence up to 98 % ("turn-off"). In general, ZnPcs@GO recovered their fluorescence ("turn-on") after the titrations and showed affinity to G4 (KD up to 1.92 µM). This is the first report that highlights the contribution of GO interfaces to assemble ZnPcs and allow their slow and controlled release to detect G4 over longer periods.

Colloidal nanomaterials for water quality improvement and monitoring.

Water is the most important resource for all kind forms of live. It is a vital resource distributed unequally across different regions of the globe, with populations already living with water scarcity, a situation that is spreading due to the impact of climate change. The reversal of this tendency and the mitigation of its disastrous consequences is a global challenge posed to Humanity, with the scientific community assuming a major obligation for providing solutions based on scientific knowledge. This article reviews literature concerning the development of nanomaterials for water purification technologies, including collaborative scientific research carried out in our laboratory (nanoLAB@UA) framed by the general activities carried out at the CICECO-Aveiro Institute of Materials. Our research carried out in this specific context has been mainly focused on the synthesis and surface chemical modification of nanomaterials, typically of a colloidal nature, as well as on the evaluation of the relevant properties that arise from the envisaged applications of the materials. As such, the research reviewed here has been guided along three thematic lines: 1) magnetic nanosorbents for water treatment technologies, namely by using biocomposites and graphite-like nanoplatelets; 2) nanocomposites for photocatalysis (e.g., TiO2/Fe3O4 and POM supported graphene oxide photocatalysts; photoactive membranes) and 3) nanostructured substrates for contaminant detection using surface enhanced Raman scattering (SERS), namely polymers loaded with Ag/Au colloids and magneto-plasmonic nanostructures. This research is motivated by the firm believe that these nanomaterials have potential for contributing to the solution of environmental problems and, conversely, will not be part of the problem. Therefore, assessment of the impact of nanoengineered materials on eco-systems is important and research in this area has also been developed by collaborative projects involving experts in nanotoxicity. The above topics are reviewed here by presenting a brief conceptual framework together with illustrative case studies, in some cases with original research results, mainly focusing on the chemistry of the nanomaterials investigated for target applications. Finally, near-future developments in this research area are put in perspective, forecasting realistic solutions for the application of colloidal nanoparticles in water cleaning technologies.

Biomimetic Graphene/Spongin Scaffolds for Improved Osteoblasts Bioactivity via Dynamic Mechanical Stimulation.

Biomimetics offers excellent prospects for design a novel generation of improved biomaterials. Here the controlled integration of graphene oxide (GO) derivatives with a 3D marine spongin (MS) network is explored to nanoengineer novel smart bio-based constructs for bone tissue engineering. The results point out that 3D MS surfaces can be homogeneously coated by layer-by-layer (LbL) assembly of oppositely charged polyethyleneimine (PEI) and GO. Notably, the GOPEI@MS bionanocomposites present a high structural and mechanical stability under compression tests in wet conditions (shape memory). Dynamic mechanically (2 h of sinusoidal compression cyclic interval (0.5 Hz, 0-10% strain)/14 d) stimulates GOPEI@MS seeded with osteoblast (MC3T3-E1), shows a significant improvement in bioactivity, with cell proliferation being two times higher than under static conditions. Besides, the dynamic assays show that GOPEI@MS bionanocomposites are able to act as mechanical stimulus-responsive scaffolds able to resemble physiological bone extracellular matrix (ECM) requirements by strongly triggering mineralization of the bone matrix. These results prove that the environment created by the system cell-GOPEI@MS is suitable for controlling the mechanisms regulating mechanical stimulation-induced cell proliferation for potential in vivo experimentation.

Graphene Oxide and Graphene Quantum Dots as Delivery Systems of Cationic Porphyrins: Photo-Antiproliferative Activity Evaluation towards T24 Human Bladder Cancer Cells.

The development of new photodynamic therapy (PDT) agents designed for bladder cancer (BC) treatments is of utmost importance to prevent its recurrence and progression towards more invasive forms. Here, three different porphyrinic photosensitizers (PS) (TMPyP, Zn-TMPyP, and P1-C5) were non-covalently loaded onto graphene oxide (GO) or graphene quantum dots (GQDs) in a one-step process. The cytotoxic effects of the free PS and of the corresponding hybrids were compared upon blue (BL) and red-light (RL) exposure on T24 human BC cells. In addition, intracellular reactive oxygen species (ROS) and singlet oxygen generation were measured. TMPyP and Zn-TMPyP showed higher efficiency under BL (IC50: 0.42 and 0.22 µm, respectively), while P1-C5 was more active under RL (IC50: 0.14 µm). In general, these PS could induce apoptotic cell death through lysosomes damage. The in vitro photosensitizing activity of the PS was not compromised after their immobilization onto graphene-based nanomaterials, with Zn-TMPyP@GQDs being the most promising hybrid system under RL (IC50: 0.37 µg/mL). Overall, our data confirm that GO and GQDs may represent valid platforms for PS delivery, without altering their performance for PDT on BC cells.

Shaping gold nanocomposites with tunable optical properties.

We report the synthesis of morphological uniform composites using miniemulsions of poly(tert-butyl acrylate) or poly(styrene) containing organically capped gold nanocrystals (NCs). The optical features of such hybrid structures are dominated by plasmonic effects and depend critically on the morphology of the resulting nanocomposite. In particular, we demonstrate the ability to tune the overall optical response in the visible spectral region by varying the Au NCs arrangement within the polymer matrix, and therefore the interparticle plasmon coupling, using Au NCs resulting from the same batch of synthesis. This is a consequence of two well-known effects on the optical properties of Au particles: the variation of the surrounding dielectric refractive index and interparticle plasmonic coupling. The research reported here shows a general strategy to produce optical responsive nanocomposites via control of the morphology of submicrometric polymer particles containing metal nanocrystals and thus is an alternative to the more common strategy of size tuning metal nanoparticles used as nanofillers.

Biofunctionalized magnetic hydrogel nanospheres of magnetite and kappa-carrageenan.

Magnetic hydrogel kappa-carrageenan nanospheres were successfully prepared via water-in-oil (w/o) microemulsions combined with thermally induced gelation of the polysaccharide. The size of the nanospheres (an average diameter of about 50 and 75 nm) was modulated by varying the concentration of surfactant. The nanospheres contained superparamagnetic magnetite nanoparticles (average diameter 8 nm), previously prepared by co-precipitation within the biopolymer. Carboxyl groups, at a concentration of about 4 mmol g(-1), were successfully grafted at the surface of these magnetic nanospheres via carboxymethylation of the kappa-carrageenan. The carboxylated nanospheres were shown to be thermo-sensitive in the 37-45 degrees C temperature range, indicating their potential as thermally controlled delivery systems for drugs and/or magnetic particles at physiological temperatures. Finally, preliminary results have been obtained for IgG antibody conjugation of the carboxylated nanospheres and the potential of these systems for bio-applications is discussed.

An integrated approach for trace detection of pollutants in water using polyelectrolyte functionalized magneto-plasmonic nanosorbents.

Resistance of pathogenic micro-organisms to conventional antibiotics is an essential issue for public health. The presence of such pharmaceuticals in aquatic ecosystems has been of major concern for which remediation and ultra-sensitive monitoring methods have been proposed. A less explored strategy involves the application of multifunctional nanosorbents for the uptake and subsequent detection of vestigial contaminants. In this study, colloidal nanoparticles (NPs) of iron oxide and gold were encapsulated in multi-layers of a charged polyelectrolyte (PEI: polyethyleneimine), envisaging the effective capture of tetracycline (TC) and its subsequent detection by Surface Enhanced Raman Scattering (SERS). Adsorption studies were performed by varying operational parameters, such as the solution pH and contact time, in order to evaluate the performance of the nanosorbents for the uptake of TC from water. While the magnetic nanosorbents with an external PEI layer (Fe3O4@PEI and Fe3O4@PEI-Au@PEI particles) have shown better uptake efficiency for TC, these materials showed less SERS sensitivity than the Fe3O4@PEI- Au nanosorbents, whose SERS sensitivity for TC in water has reached the limit of detection of 10 nM. Thus, this study highlights the potential of such magneto-plasmonic nanosorbents as multi-functional platforms for targeting specific contaminants in water, by taking into consideration both functionalities investigated: the removal by adsorption and the SERS detection across the nanosorbents' surfaces.

Can contaminated waters or wastewater be alternative sources for technology-critical elements? The case of removal and recovery of lanthanides.

Technology critical elements (TCE) are considered the vitamins of nowadays technology. Factors such as high demand, limited sources and geopolitical pressures, mining exploitation and its negative impact, point these elements as new emerging contaminants and highlight the importance for removal and recycling TCE from contaminated waters. This paper reports the synthesis, characterization and application of hybrid nanostructures to remove and recover lanthanides from water, promoting the recycling of these high value elements. The nanocomposite combines the interesting properties of graphite nanoplatelets, with the magnetic properties of magnetite, and exhibits good sorption properties towards La(III), Eu(III) and Tb(III). The sorption process was very sensitive to solution pH, evidencing that electrostatic interactions are the main binding mechanism involved. Removal efficiencies up to 80% were achieved at pH 8, using only 50 mg/L of nanocomposite. In ternary solution, occurred a preferential removal of Eu(III) and Tb(III). The equilibrium evidenced a rare but interesting behaviour, and as a proof-of-concept the recoveries and reutilization rates, at consecutive cycles, highlight the recyclability of the composite without loss of efficiency. This study evidences that surface charge and the number of active sites of the composite controls the removal process, providing new insights on the interactions between lanthanoids and magnetic-graphite-nanoplatelets.

Novel sintering-free scaffolds obtained by additive manufacturing for concurrent bone regeneration and drug delivery: Proof of concept.

Advances on the fabrication of sintering-free biphasic calcium phosphate (BCP)/natural polymer composite scaffolds using robocasting as additive manufacturing technique are presented in the present work. Inks with high amounts of BCP powders (45 vol%) containing different HA/ß-TCP ratios, in presence of crosslinked polymer, were successfully fine-tuned for extrusion by robocasting. The non-existence of sintering step opened the possibility to obtain drug loaded scaffolds by adding levofloxacin to the extrudable inks. The drug presence induced slightly changes on the rheological behaviour of the inks, more emphasized for the BCP compositions with higher amounts of ß-TCP, and consequently, on the microstructure and on the mechanical properties of the final scaffolds. The strong interaction of ß-TCP with chitosan difficult the preparation of suitable rheological inks for printing. Drug delivery studies revealed a fast release of levofloxacin with a high burst of drug within the first 30 min. Levofloxacin loaded samples also presented bacteria growth inhibition ability, proving that antibiotic was not degraded during the fabrication process and its bactericidal efficacy was preserved. From the results obtained, the composite scaffolds containing higher amounts of HA (around 80% HA/20% ß-TCP) constitute a promising bi-functional synthetic bone substitute for simultaneous local bone regeneration and infection treatments.