BioMIPs: molecularly imprinted silk fibroin nanoparticles to recognize the iron regulating hormone hepcidin

: The possibility to prepare molecularly imprinted nanoparticles from silk fibroin was recently demonstrated starting from methacrylated silk fibroin and choosing a protein as template. Here, we attempted the imprinting of fibroin-based molecularly imprinted polymers (MIPs), called bioMIPs, using as a template hepcidin that is a iron-metabolism regulator-peptide, possessing a hairpin structure. A homogeneous population (PDI < 0.2) of bioMIPs with size ~50 nm was produced. The bioMIPs were selective for the template; the estimated dissociation constant for hepcidin was KD = 3.6 ± 0.5 10-7 M and the average number of binding sites per bioMIP was equal to 2. The bioMIPs used in a competitive assay for hepcidin in serum showed a detection range of 1.01 10-7- 6.82 10-7 M and a limit of detection of 3.29 10-8 M

Silk fibroin molecularly imprinted nanoparticles as biocompatible molecular nanotraps: Molecular recognition ties the knot with biomaterials. The bioMIP’s labeling and degradation

Molecularly imprinted nanoparticles (nanoMIPs) are biomimetic polymeric nanomaterials, typically prepared from acrylamide and derivatives, that are formed by a template-assisted synthesis. NanoMIPs display high afnity, selectivity, and specifcity for the targeted molecule, on the par of natural receptors and antibodies. Recently, we introduced a paradigmatic change by forming nanoMIPs starting from biomaterials, under the name of bioMIPs, as a strategy to promptly translate them into the clinical settings. Silk fbroin, that is a biocompatible and non-immunogenic natural material, was used as a building block for the synthesis of bioMIPs tailored to recognize the protein human serum albumin. BioMIPs confrmed high selectivity and specifcity for the targeted protein, together with cytocompatibility. The present work expands the actual knowledge on bioMIPs, studying a route to post-synthetically entail fuorescent tags, with the aim to localize these molecular nanotraps in cells and tissues. Moreover, the enzymatic degradation of bioMIPs was investigated, to support the role of bioMIPs as greener and biocompatible alternatives to non-natural biomimetics

Time-Resolved Fluorescence Spectroscopy of Molecularly Imprinted Nanoprobes as an Ultralow Detection Nanosensing Tool for Protein Contaminants

: Currently, optical sensors based on molecularly imprinted polymers (MIPs) have been attracting significant interest. MIP sensing relies on the combination of the MIP's selective capability, which is conveyed to the polymeric material by a template-assisted synthesis, with optical techniques that offer exquisite sensitivity. In this work, we devised an MIP nanoparticle optical sensor for the ultralow detection of serum albumin through time-resolved fluorescence spectroscopy. The Fluo-nanoMIPs (∅~120 nm) were synthetized using fluorescein-O-methacrylate (0.1×, 1×, 10× mol:mol versus template) as an organic fluorescent reporter. The ability of 0.1× and 1×Fluo-nanoMIPs to bind albumin (15 fM-150 nM) was confirmed by fluorescence intensity analyses and isothermal titration calorimetry. The apparent dissociation constant (Kapp) was 30 pM. Conversely, the 10× fluorophore content did not enable monitoring binding. Then, the time-resolved fluorescence spectroscopy of the nanosensors was studied. The 1×Fluo-nanoMIPs showed a decrease in fluorescence lifetime upon binding to albumin (100 fM-150 nM), Kapp = 28 pM, linear dynamic range 3.0-83.5 pM, limit of detection (LOD) 1.26 pM. Selectivity was confirmed testing 1×Fluo-nanoMIPs against competitor proteins. Finally, as a proof of concept, the nanosensors demonstrated detection of the albumin (1.5 nM) spiked in wine samples, suggesting a possible scaling up of the method in monitoring allergens in wines

On the Effect of Soft Molecularly Imprinted Nanoparticles Receptors Combined to Nanoplasmonic Probes for Biomedical Applications

Soft, deformable, molecularly imprinted nanoparticles (nanoMIPs) were combined to nano-plasmonic sensor chips realized on poly (methyl methacrylate) (PMMA) substrates to develop highly sensitive bio/chemical sensors. NanoMIPs (d(mean) < 50 nm), which are tailor-made nanoreceptors prepared by a template assisted synthesis, were made selective to bind Bovine Serum Albumin (BSA), and were herein used to functionalize gold optical nanostructures placed on a PMMA substrate, this latter acting as a slab waveguide. We compared nanoMIP-functionalized non-optimized gold nanogratings based on periodic nano-stripes to optimized nanogratings with a deposited ultra-thin MIP layer (<100 nm). The sensors performances were tested by the detection of BSA using the same setup, in which both chips were considered as slab waveguides, with the periodic nano-stripes allocated in a longitudinal orientation with respect to the direction of the input light. Result demonstrated the nanoMIP-non optimized nanogratings showed superior performance with respect to the ultra-thin MIP-optimized nanogratings. The peculiar deformable character of the nano-MIPs enabled to significantly enhance the limit of detection (LOD) of the plasmonic bio/sensor, allowing the detection of the low femtomolar concentration of analyte (LOD similar to 3 fM), thus outpassing of four orders of magnitude the sensitivies achieved so far on optimized nano-patterned plasmonic platforms functionalized with ultra-thin MIP layers. Thus, deformable nanoMIPs onto non-optimized plasmonic probes permit to attain ultralow detections, down to the quasi-single molecule. As a general consideration, the combination of more plasmonic transducers to different kinds of MIP receptors is discussed as a mean to attain the detection range for the selected application field

Non-Specific Responsive Nanogels and Plasmonics to Design MathMaterial Sensing Interfaces: The Case of a Solvent Sensor

: The combination of non-specific deformable nanogels and plasmonic optical probes provides an innovative solution for specific sensing using a generalistic recognition layer. Soft polyacrylamide nanogels that lack specific selectivity but are characterized by responsive behavior, i.e., shrinking and swelling dependent on the surrounding environment, were grafted to a gold plasmonic D-shaped plastic optical fiber (POF) probe. The nanogel-POF cyclically challenged with water or alcoholic solutions optically reported the reversible solvent-to-phase transitions of the nanomaterial, embodying a primary optical switch. Additionally, the non-specific nanogel-POF interface exhibited more degrees of freedom through which specific sensing was enabled. The real-time monitoring of the refractive index variations due to the time-related volume-to-phase transition effects of the nanogels enabled us to determine the environment's characteristics and broadly classify solvents. Hence the nanogel-POF interface was a descriptor of mathematical functions for substance identification and classification processes. These results epitomize the concept of responsive non-specific nanomaterials to perform a multiparametric description of the environment, offering a specific set of features for the processing stage and particularly suitable for machine and deep learning. Thus, soft MathMaterial interfaces provide the ground to devise devices suitable for the next generation of smart intelligent sensing processes

Methacrylated Silk Fibroin Additive Manufacturing of Shape Memory Constructs with Possible Application in Bone Regeneration

: Methacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications

Soft molecularly imprinted nanoparticles with simultaneous lossy mode and surface plasmon multi-resonances for femtomolar sensing of serum transferrin protein

: The simultaneous interrogation of both lossy mode (LMR) and surface plasmon (SPR) resonances was herein exploited for the first time to devise a sensor in combination with soft molecularly imprinting of nanoparticles (nanoMIPs), specifically entailed of the selectivity towards the protein biomarker human serum transferrin (HTR). Two distinct metal-oxide bilayers, i.e. TiO2-ZrO2 and ZrO2-TiO2, were used in the SPR-LMR sensing platforms. The responses to binding of the target protein HTR of both sensing configurations (TiO2-ZrO2-Au-nanoMIPs, ZrO2-TiO2-Au-nanoMIPs) showed femtomolar HTR detection, LODs of tens of fM and KDapp ~ 30 fM. Selectivity for HTR was demonstrated. The SPR interrogation was more efficient for the ZrO2-TiO2-Au-nanoMIPs configuration (sensitivity at low concentrations, S = 0.108 nm/fM) than for the TiO2-ZrO2-Au-nanoMIPs one (S = 0.061 nm/fM); while LMR was more efficient for TiO2-ZrO2-Au-nanoMIPs (S = 0.396 nm/fM) than for ZrO2-TiO2-Au-nanoMIPs (S = 0.177 nm/fM). The simultaneous resonance monitoring is advantageous for point of care determinations, both in terms of measurement's redundancy, that enables the cross-control of the measure and the optimization of the detection, by exploiting the individual characteristics of each resonance

Soluble collagen dissolution and assembling in pressurized carbon dioxide water solutions

Dissolution and gelation procedures have a great influence on gelation time, microstructure and mechanical properties of reconstituted collagen products. We have investigated the dissolution of atelocollagen in CO2/water solutions at low temperature (4 degrees C) at different CO2 pressures (0.3-0.9 MPa), as well as gelation kinetics and physico-chemical properties of the hydrogel obtained after CO2 removal. Compared to conventional methods, the CO2-assisted technique resulted in faster soluble collagen dissolution and faster gelation into transparent gels characterized by thin 10 nm fibrils. Electrophoresis and CD spectroscopy demonstrated that the process did not denature the soluble collagen. The possibility to obtain collagen dissolution and gelation without the use of chemical agent other than water and CO2 makes this process particularly appealing for biomedical applications

Antibacterial properties of photo-crosslinked chitosan/methacrylated hyaluronic acid nanoparticles loaded with bacitracin

The current treatments for wounds often fail to produce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20⠯%). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478⠯nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742⠯nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332⠯nm. The encapsulation of BC was highly efficient (97⠯%). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. The nanoparticles loaded with BC based on photo-crosslinked HAMA/CS demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion