Bone formation requires a substantial energy supply to sustain extracellular matrix production and mineralization, yet the temporal contribution of lipid metabolism during osteoblast maturation remains incompletely characterized. This study investigated the molecular and transcriptional remodeling of lipid metabolism. Intracellular lipid distribution was analyzed by confocal microscopy using Nile Red staining. Transcriptional modulation of lipid synthesis, storage, lipolysis, genes associated with mitochondrial fatty acid oxidation, and osteogenic markers were assessed by quantitative real-time PCR, and the biochemical composition was evaluated by Raman spectroscopy. Early stages of spheroid development showed higher expression of genes involved in lipid synthesis and storage (FASN, DGAT2, and PLIN2) together with intracellular lipid accumulation, whereas later stages displayed increased expression of lipolytic and β-oxidation markers (PNPLA2/ATGL, CPT1A, and HADHA), accompanied by the redistribution of lipid droplets. The Raman analysis revealed a time-dependent variation of lipid-associated CH2/CH3 bands and modulation of protein-related Amide I-III signals, consistent with biochemical remodeling during maturation. Overall, the data indicate a coordinated transcriptional shift from lipid accumulation-associated pathways toward lipid mobilization during osteogenic progression in a 3D culture. This model provides a controlled experimental platform for investigating metabolic regulation during bone formation and for studying metabolic alterations associated with skeletal disorders.

ABSTRACT Electrochemiluminescence (ECL) biosensing offers high sensitivity and low background but remains limited by the lack of simple strategies for electrode patterning, signal confinement, and multiplexed detection. Here, we report a silica nanoparticle–based coating and photopatterning approach for gold electrodes that enables spatially resolved and multiplexed ECL detection. The resulting electrode architecture confines aqueous droplets to designated locations and supports localized ECL signal generation. Compared with bare gold electrodes, the modified surfaces exhibit up to a ten‐fold increase in ECL intensity, which correlates with reduced charge‐transfer resistance and improved electron‐transfer kinetics. The patterned electrodes are compatible with magnetic bead–based immunoassays and allow parallel ECL measurements from multiple spatially separated regions on a single chip. Multiplexed detection is demonstrated using the synthetic SARS‐CoV‐2 spike protein as a model analyte. This coating and patterning strategy provides a straightforward route to spatially resolved ECL electrodes and can be applied to multiplexed electrochemical and bioanalytical measurements where signal confinement and multiplexing are required.

The technological evolution towards portable systems for nucleic acids (NAs) analysis is central in diagnostics and healthcare. Conventional approaches for genetic analysis require multistep procedures, from the genetic material purification to its detection, that are mostly based on the NA amplification by polymerase chain reaction (PCR) and constrained to bulky specialized instruments and dedicated laboratories. These limitations imply an increase in time and cost of analysis avoiding the possibility of massive molecular screening, as in the case of an infectious pandemic, and accessible diagnosis. Therefore, research efforts are moving towards new Lab-on-a-chip (LoC) technologies that guarantee a more integrated, decentralized, user-friendly, and low-cost NA analysis. These are based on miniaturized optical or electrical chips on which it is possible to integrate a sensing surface for specific analytical applications. In this scenario, we presented the fabrication and characterization of a miniaturized electrochemical (EC) biochip as a potential LoC for genetic applications. The chip was developed with microfabricated electrodes on top and polycarbonate/PDMS microchambers for the surface treatment and functionalization. The characterization of chip EC performances has been performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the chip surface has been functionalized with thiol-modified oligonucleotides, whose immobilization has been characterized by contact angle (CA) and surface free energy (SFE) analysis. Results reported a good robustness and responsivity of the chip proving its suitability for sensing array preparation and integration in a LoC format.

Nanomaterials represents a class of multifunctional materials that can play a key role in multiple applications of biomedicine. In case of severe bone defects due to trauma, cancer or aging, a huge demand for more effective treatments is one of the important challenges in clinical practice. Biomaterials for bone tissue engineering (BTE) is one of the appealing approach to enhance regenerative capability by combining several biocomponents like biomimetic materials and nanomaterials, cells, growth factors. Hydroxyapatite (HA) represents the most used based biomaterial for bone regeneration. However, there is still a crucial demand for new HA-based biomaterials satisfying clinical needs in terms of improved osteointegration and osteoregeneration. Here we show the synergic application of two nanomaterials, i.e., a fluorogenic hyaluronan nanogel (hyaluronic acid with Rhodamine B, HyRB) and a class of theranostic core–shell silica nanoparticles (PluSNPs) integrated into MgHA-type I collagen-based scaffolds. This novel biomaterial (HyRB/PluSNPs doped MgHA) showed improved performance in osteoregeneration, both for osteoconductivity and osteoinductivity; furthermore, the HyRB/PluSNPs nanomaterials can act as contrast agent for fluorescence imaging and have a potential for targeted drug delivery and for phototherapy. Results pave the way for the development of new multifunctional biomaterials combining the advantages of improved osteoconductivity/osteoinductivity of HyRB/PluSNPs with their potential drug delivery functionality and phototherapeutic properties.

Cell-cell communication and extracellular matrix (ECM) organization in a bone microenvironment are essential to replicate the bone microenvironment accurately. In this study, the extracellular matrix (ECM) was emulated by incorporating M13 phages, selected through phage display for displaying engineered peptides that mimic bone matrix proteins, into human osteoblast cultures to develop a three-dimensional bone model (3D BMP-Phage). Comprehensive analysis was performed to investigate: (i) the morphological development of spheroids, assessed by optical microscopy and quantified via fractal dimension analysis using box-counting algorithms; (ii) the biochemical composition of the extracellular matrix, evaluated by Raman spectroscopy; (iii) ECM protein deposition, analyzed through immunofluorescence staining; (iv) matrix mineralization, assessed by Alizarin Red staining and alkaline phosphatase (ALP) activity assay; and (v) osteogenic gene expression, measured by quantitative RT-PCR. The findings demonstrate that the 3D BMP-Phage model, facilitated by a cocktail of bone-mimicking peptides, enhances structural integrity, ECM complexity, mineralization, and osteogenic pathways compared to the control. This novel approach replicates key aspects of the bone microenvironment, providing a valuable platform for advanced physiological and regenerative medicine research under controlled conditions.

The emergence of antibiotic-resistant bacteria underscores the need for innovative and precise therapeutic strategies. Here, we present a novel approach to combating bacterial infections by developing engineered phage-silver nanoparticle (AgNP) complexes as targeted therapeutic agents. We first synthesized and characterized AgNPs using advanced techniques, ensuring precise particle size and surface charge control. Subsequently, we combined the AgNPs with engineered M13 bacteriophages (Li5 phage) displaying a foreign peptide that provides selectivity for specific E. coli strains. We found that the AgNP@Li5 phage molecular complex exhibited highly selective antibacterial activity against E. coli F+, F- and pathogenic O157:H7 strains while having little impact on other bacterial species (p < 0.0001). AgNPs@Li5 demonstrated antibacterial activity with similar MIC values for E. coli TG1 and E. coli F-, inhibiting bacterial growth at a 1:16 dilution. In contrast, the antibacterial activity against E. coli O157:H7 was lower, with a dilution value of 1:8, compared to the other E. coli strains. The specificity of this approach minimizes collateral damage to surrounding bacteria, addressing a key challenge in conventional antimicrobial therapies. This system can be easily customized to target pathogens and tumors by simply modifying the peptides displayed on the phages. Our findings highlight the potential for innovative approaches in targeted therapy.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide. Recent advances in organoid technology have revolutionized the in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate the cellular heterogeneity, structure, and functions of organs and tissues. In this work, 3D MicroOrganoSpheres (MOSs) were developed from human liver cancer HepG2 cell lines (HCC-MOSs) embedded in methylcellulose and dispersed in mineral oil by a 2-channels droplet generator chip controlled by two separate pumps. The HCC-MOSs have been maintained in culture and their growth and development were monitored to create a preliminary model for organoids. MOSs on-chip can be used to simulate the complex structure, microenvironment, and biological functions of HCC and provide an in vitro model to study the physiological processes underlying the tumor and the response to therapeutic treatment.

Abstract Alzheimer's disease (AD) is a diffused neurodegenerative disorder affecting people in advanced age causing loss of memory and dementia. Nowadays, diagnosis and treatment of AD are still challenging due to the lack of diagnostic systems that allow for an early and reliable diagnosis and therapy monitoring. Moreover, conventional strategies for AD diagnosis are based on brain imaging techniques that are invasive and expensive for early and massive screening. Phage display approach, using engineered phage probe for direct amyloid‐β (Aβ)‐autoantibodies detection, overcome these limitations leading to the possibility of safe and low‐cost screening. Moreover, the combination with silicon technology further improves the easiness of diagnosis due to the portability of devices and the integration of sensitive transduction signals. In this work, an innovative silicon‐based sensing technology is reported detecting Aβ‐autoantibodies, specifically Immunoglobulin G (IgG), in human sera by engineered M13‐phage probes (ADPP). The strategy hinges on a bio‐surface that is integrated on top of a silicon biosensor. Thanks to phages probes exposing Aβ‐mimic peptides, this chip can capture and reveal Aβ‐autoantibodies, discriminating between healthy and AD conditions. The surface chemistry is morphologically and chemically characterized and the phage‐based biosensor ability to recognise Aβ‐autoantibodies is proved by transduction with enzyme‐linked anti‐M13 antibodies.

Silicon nanowires (Si NWs) are considered an outstanding material for several applications. We have realized quantum-confined and room-temperature luminescent Si NWs. These Si NWs exhibit a high-aspect ratio, and high sensitive and selective label-free detection has been demonstrated for proteins, small extracellular vesicles, and DNAs. The realization of a SARS-CoV-2 Si NW sensor able to detect a few virus copies and remain unaffected by the variant (such as Omicron) is reported, paving the way for new, cheap, optical label-free devices for the primary health care diagnosis with an industrially compatible approach.

An innovative biosensing strategy for the diagnosis of Alzheimer’s disease (AD) in human sera has been developed. The technology relied on a silicon flat substrate that was functionalized to perform a phage display detection of anti-amyloid beta (Aβ) antibodies, as AD markers, among the pool of IgGs of human sera. The substrate was derivatized with an interface able to bind and orient the IgGs for the detection operated by an engineered selective probe phage. The interface chemistry and its discrimination activity of healthy and AD sera have been fully characterized.

AbstractPCR‐free approaches are the most promising technologies for molecular point‐of‐care (PoC). In this context, the detection of not amplified genetic targets through electro‐optical transduction is successfully investigated. While PCR‐free approaches are widely studied, there are only a few studies investigating the factors that modulate both the kinetics and the effectiveness of target capture. Among these, the probes grafting density and the isoelectric properties of the biointerface are crucial since they conditionate the charge field around biomolecules during and after the target recognition. In this work, an experimental and theoretical study of a gold biointerface functionalized with oligonucleotide probes is presented for the direct detection by cooperative hybridization of the kinetoplast (k)DNA of Leishmania infantum(LI). The biointerface is characterized by surface free energy (SFE) analysis and contact angle (CA) to investigate the grafting of probes and the surface isoelectric properties upon the duplex formation with the genetic target. Experimental data are compared with a theoretical model, based on the prediction of adsorption energies, which effectively reflects the charge profile of the functionalized surface. Lastly, the biointerface is characterized by electrochemical impedance spectroscopy (EIS) and the sensing performances assess in the frame of its suitability for PoC applications.

55 million people worldwide suffer from Alzheimer's disease (AD). A definitive diagnosis of AD is made postmortem after a neuropathological examination of the brain. There is an urgent need for an innovative, noninvasive methodology that allows for an early and reliable diagnosis. Several engineered phages that recognized Aβ-autoantibodies present in the sera of AD patients are previously identified. Here, novel phages are tested for their ability to accurately discriminate AD sera using immunophage-polymerase chain reaction in a miniatured biochip. It is found that five of the six phages analyzed discriminate between healthy controls and AD patients. Further, by combining the response of two phages, non-AD and severe AD cases are identified with 100% accuracy and mild-to-moderate cases with 90% accuracy. While the number of cases used here are relatively small and can be confirmed in larger cohorts, this first-of-a-kind system represents an innovative methodology with the potential of having a major impact in the AD field: from a clinical perspective, it can aid physicians in making an accurate AD diagnosis; from a research perspective, it can be used as a surrogate for AD clinical trials.

Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field.

The pandemic outbreak caused by SARS-CoV-2 coronavirus brought a crucial issue in public health causing up to now more than 600 million infected people and 6.5 million deaths. Conventional diagnostic methods are based on quantitative reverse transcription polymerase chain reaction (RT-qPCR assay) and immuno-detection (ELISA assay). However, despite these techniques have the advantages of being standardized and consolidated, they keep some main limitations in terms of accuracy (immunoassays), time/cost consumption of analysis, the need for qualified personnel, and lab constrain (molecular assays). There is crucial the need to develop new diagnostic approaches for accurate, fast and portable viral detection and quantification. Among these, PCR-free biosensors represent the most appealing solution since they can allow molecular detection without the complexity of the PCR. This will enable the possibility to be integrated in portable and low-cost systems for massive and decentralized screening of SARS-CoV-2 in a point-of-care (PoC) format, pointing to achieve a performant identification and control of infection. In this review, the most recent approaches for the SARS-CoV-2 PCR-free detection are reported, describing both the instrumental and methodological features, and highlighting their suitability for a PoC application.

Abstract The recent SARS‐CoV‐2 pandemic has highlighted the urgent need for novel point‐of‐care devices to be promptly used for a rapid and reliable large screening analysis of several biomarkers like genetic sequences and antibodies. Currently, one of the main limitations of rapid tests is the high percentage of false negatives in the presence of variants and, in particular for the Omicron one. We demonstrate in this work the detection of SARS‐CoV‐2 and the Omicron variant with a cost‐effective silicon nanosensor enabling high sensitivity, selectivity, and fast response. We have shown that a silicon (Si) nanowires (NW) platform detects both Sars‐CoV‐2 and its Omicron variant with a limit of detection (LoD) of four effective copies (cps), without any amplification of the genome, and with high selectivity. This ultrasensitive detection of 4 cps allows to obtain an extremely early diagnosis paving the way for efficient and widespread tracking. The sensor is made with industrially compatible techniques, which in perspective may allow easy and cost‐effective industrialization.

The arising of new physical phenomena at the nanoscale promoted for the scientific community the emerging of silicon nanostructures for future challenging technologies involving innovative applications in light management and photonics. Silicon nanowires (Si NWs) are already considered strategic systems for very different applications such as microelectronics, energy, and sensors. Nonetheless, the use of Si NWs for photonic applications is very limited. The reason for these poor results in the photonic field is related to the fabrication methods that generally are used to obtain silicon nanowires. One of the most promising approaches to realize Si NWs with a low-cost and Si technology compatibility is metal-assisted chemical etching. In this paper, we report a review of silicon nanowires realized by metal chemical etching for photonic applications focusing our attention on the realization of light-emitting Si NWs.

Carbon nanomaterials have shown great potential in several fields, including biosensing, bioimaging, drug delivery, energy, catalysis, diagnostics, and nanomedicine. Recently, a new class of carbon nanomaterials, carbon dots (CDs), have attracted much attention due to their easy and inexpensive synthesis from a wide range of precursors and fascinating physical, chemical, and biological properties. In this work we have developed CDs derived from olive solid wastes of two Mediterranean regions, Puglia (CDs_P) and Calabria (CDs_C) and evaluated them in terms of their physicochemical properties and antibacterial activity against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Results show the nanosystems have a quasi-spherical shape of 12–18 nm in size for CDs_P and 15–20 nm in size for CDs_C. UV–Vis characterization indicates a broad absorption band with two main peaks at about 270 nm and 300 nm, respectively, attributed to the π-π* and n-π* transitions of the CDs, respectively. Both samples show photoluminescence (PL) spectra excitation-dependent with a maximum at λem = 420 nm (λexc = 300 nm) for CDs_P and a red-shifted at λem = 445 nm (λexc = 300 nm) for CDs_C. Band gaps values of ≈ 1.48 eV for CDs_P and ≈ 1.53 eV for CDs_C are in agreement with semiconductor behaviour. ζ potential measures show very negative values for CDs_C compared to CDs_P (three times higher, −38 mV vs. −18 mV at pH = 7). The evaluation of the antibacterial properties highlights that both CDs have higher antibacterial activity towards Gram-positive than to Gram-negative bacteria. In addition, CDs_C exhibit bactericidal behaviour at concentrations of 360, 240, and 120 µg/mL, while lesser activity was found for CDs_P (bacterial cell reduction of only 30% at the highest concentration of 360 µg/mL). This finding was correlated to the higher surface charge of CDs_C compared to CDs_P. Further investigations are in progress to confirm this hypothesis and to gain insight on the antibacterial mechanism of both cultivars.

Legionella pneumophila contamination of water systems is a crucial issue for public health. The pathogen is able to persist in water as free-living planktonic bacteria or to grow within biofilms that adhere to and clog filters and pipes in a water system, reducing its lifespan and, in the case of hospital buildings, increasing the risk of nosocomial infections. The implementation of water management is considered to be the main prevention measure and can be achieved from the optimization of water system architecture, notably introducing new materials and strategies to contrast Legionella biofilm proliferation and so prolong the water system functionality. In this research, we propose a new smart surface against L. pneumophila biofilm formation. This is based on an innovative type of coating consisting of a sulfonated pentablock copolymer (s-PBC, commercially named Nexar™) deposited on top of a polypropylene (PP) coupon in a sandwich filter model. The covering of PP with s-PBC results in a more hydrophilic, acid, and negatively charged surface that induces microbial physiological inhibition thereby preventing adhesion and/or proliferation attempts of L. pneumophila prior to the biofilm formation. The antibiofilm property has been investigated by a Zone of Inhibition test and an in vitro biofilm formation analysis. Filtration tests have been performed as representative of possible applications for s-PBC coating. Results are reported and discussed.

The past few decades have seen rapid advances in surface-enhanced Raman scattering (SERS) techniques which have enabled increasingly efficient detection of infinitesimal molecular traces down to the single molecule. This occurrence paved the way for promising applications, among which the use of SERS in highly efficient label-free optical sensors, thus attaining a large interest, especially in the biomedical field. SERS sensors are realized following different strategies, usually involving the control of size and shape of nanoantennas and the design of nanostructures arrays to precisely set the Localized Surface Plasmon resonances (LSP) and the hot spots positions. However, this may entail the use of lithographic techniques with high fabrication costs. Other approaches are based on disordered arrays of plasmonic nanostructures, much cheaper and easier to fabricate, although the control of the hot spots position and performances is lost. Here we review the more recent SERS strategies approached in order to obtain the desired detection of analytes and specific biomolecular species, ranging from the realization of particularly efficient SERS substrates suitable for hydrated molecules to the creation of hot spots in a liquid environment enabling highly sensitive spectroscopic sensors in the natural habitat of the biomolecules.

The continuing accumulation of mutations in the RNA genome of the SARS-CoV-2 virus generates an endless succession of highly contagious variants that cause concern around the world due to their antibody resistance and the failure of current diagnostic techniques to detect them in a timely manner. Raman spectroscopy represents a promising alternative to variants detection and recognition techniques, thanks to its ability to provide a characteristic spectral fingerprint of the biological samples examined under all circumstances. In this work we exploit the surface-enhanced Raman scattering (SERS) properties of a silver dendrite layer to explore, for the first time to our knowledge, the distinctive features of the Omicron variant genome. We obtain a complex spectral signal of the Omicron variant genome where the fingerprints of nucleobases in nucleosides are clearly unveiled and assigned in detail. Furthermore, the fractal SERS layer offers the presence of confined spatial regions in which the analyte remains trapped under hydration conditions. This opens up the prospects for a prompt spectral identification of the genome in its physiological habitat and for a study on its activity and variability.