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.

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.

Molecularly imprinted polymers by epitope imprinting: a journey from molecular interactions to the available bioinformatics resources to scout for epitope templates.

The molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein's structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein's surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates.

A Surface Plasmon Resonance Plastic Optical Fiber Biosensor for the Detection of Pancreatic Amylase in Surgically-Placed Drain Effluent.

Postoperative pancreatic fistula (POPF), the major driver of morbidity and mortality following pancreatectomy, is caused by an abnormal communication between the pancreatic ductal epithelium and another epithelial surface containing pancreas-derived, enzyme-rich fluid. There is a strong correlation between the amylase content in surgically-placed drains early in the postoperative course and the development of POPF. A simple and cheap method to determine the amylase content from the drain effluent has been eagerly advocated. Here, we developed an amylase optical biosensor, based on a surface plasmon resonance (SPR) plastic optical fiber (POF), metallized with a 60 nm layer of gold and interrogated with white light. The sensor was made specific by coupling it with an anti-amylase antibody. Each surface derivatization step was optimized and studied by XPS, contact angle, and fluorescence. The POF-biosensor was tested for its response to amylase in diluted drain effluents. The volume of sample required was 50 µL and the measurement time was 8 min. The POF-biosensor showed selectivity for amylase, a calibration curve log-linear in the range of 0.8-25.8 U/L and a limit of detection (LOD) of ~0.5 U/L. In preliminary tests, the POF-biosensor allowed for the measurement of the amylase content of diluted surgically-placed drain effluents with an accuracy of >92% with respect to the gold standard. The POF-biosensor allows for reliable measurement and could be implemented to allow for a rapid bedside assessment of amylase value in drains following pancreatectomy.

Tailoring a Dress to Single Protein Molecules: Proteins Can Do It Themselves through Localized Photo-Polymerization and Molecular Imprinting.

Molecularly imprinted polymer nanoparticles (MIP NPs) are antibody-like recognition materials prepared by a template-assisted synthesis. MIP NPs able to target biomolecules, like proteins, are under the spotlight for their great potential in medicine, but efficiently imprinting biological templates is still very challenging. Here we propose generating a molecular imprint in single NPs, by photochemically initiating the polymerization from individual protein templates. In this way, each protein molecule tailors itself its own "polymeric dress". For this, the template protein is covalently coupled with a photoinitiator, Eosin Y. Irradiated with light at 533 nm, the Eosin moiety acts as an antenna and transfers energy to a co-initiator (an amine), which generates a radical and initiates polymerization. As a result, a polymer network is forming only around the very template molecule, producing cross-linked NPs of 50 nm, with single binding sites showing high affinity (KD 10-9 m) for their biological target, and selectivity over other proteins.

Molecular Imprinted Polymers Coupled to Photonic Structures in Biosensors: The State of Art.

Optical sensing, taking advantage of the variety of available optical structures, is a rapidly expanding area. Over recent years, whispering gallery mode resonators, photonic crystals, optical waveguides, optical fibers and surface plasmon resonance have been exploited to devise different optical sensing configurations. In the present review, we report on the state of the art of optical sensing devices based on the aforementioned optical structures and on synthetic receptors prepared by means of the molecular imprinting technology. Molecularly imprinted polymers (MIPs) are polymeric receptors, cheap and robust, with high affinity and selectivity, prepared by a template assisted synthesis. The state of the art of the MIP functionalized optical structures is critically discussed, highlighting the key progresses that enabled the achievement of improved sensing performances, the merits and the limits both in MIP synthetic strategies and in MIP coupling.

Selective PQQPFPQQ Gluten Epitope Chemical Sensor with a Molecularly Imprinted Polymer Recognition Unit and an Extended-Gate Field-Effect Transistor Transduction Unit.

A molecularly imprinted polymer (MIP) recognition system was devised for selective determination of an immunogenic gluten octamer epitope, PQQPFPQQ. For that, a thin MIP film was devised, guided by density functional theory calculations, and then synthesized to become the chemosensor recognition unit. Bis(bithiophene)-based cross-linking and functional monomers were used for this synthesis. An extended-gate field-effect transistor (EG-FET) was used as the transduction unit. The EG-FET gate surface was coated with the PQQPFPQQ-templated MIP film, by electropolymerization, to result in a complete chemosensor. X-ray photoelectron spectroscopy analysis confirmed the presence of the PQQPFPQQ epitope, and its removal from the MIP film. The chemosensor selectively discriminated between the octamer analyte and another peptide of the same number of amino acids but with two of them mismatched (PQQQFPPQ). The chemosensor was validated with respect to both the PQQPFPQQ analyte and a real gluten extract from semolina flour. It was capable to determine PQQPFPQQ in the concentration range of 0.5-45 ppm with the limit of detection (LOD) = 0.11 ppm. Moreover, it was capable of determining gluten in real samples in the concentration range of 4-25 ppm with LOD = 4 ppm, which is a value sufficient for discriminating between gluten-free and non-gluten-free food products. The gluten content in semolina flour determined with the chemosensor well correlated with that determined with a commercial ELISA gluten kit. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the epitope sorption data. The sorption parameters determined from these isotherms indicated that the imprinted cavities were quite homogeneous and that the epitope analyte was chemisorbed in them.

A microRNA signature from serum exosomes of patients with glioma as complementary diagnostic biomarker.

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.

Micro- versus nano-sized molecularly imprinted polymers in MALDI-TOF mass spectrometry analysis of peptides.

The integration of molecularly imprinted polymers (MIPs) with MALDI-TOF mass spectrometry (MS) combines MIP selectivity with MS sensitivity. Whether the size of the MIP material-micro versus nano-has an effect on the MS analysis was the object of the study. MIPs, targeting respectively the epitope peptide NR11 of cardiac troponin I and the peptide CK13 of human serum transferrin, were synthesized and characterized. The size-related performance of the MIP materials hyphenated with MALDI-TOF-MS analysis was studied by the incubation of the target peptide with the respective micro- or nano-MIP, followed by rinsing to remove non-specific deposition of the MIP to the MALDI target plate, co-crystallization with the organic matrix, and mass analysis. The quality of the MS analysis was assessed comparing the S/N of the mass peaks of the MIP-bound peptide to that of the same quantity of free peptide. Sweet spots and lower S/N (~ 1 order of magnitude) were observed for micro-MIP materials, while in the case of nano-MIP-bound peptide, the S/N was comparable to that of the free peptide, indicating higher compatibility of the nano-MIPs to MALDI-TOF-MS. The nano-MIP/MALDI-TOF-MS permitted the selective determination of the target peptide in real serum samples. Graphical abstract ᅟ.

Molecularly imprinted polymers coupled to matrix assisted laser desorption ionization mass spectrometry for femtomoles detection of cardiac troponin I peptides.

Molecularly imprinted polymers (MIPs) were combined to MALDI-TOF-MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7-2µm), dissociation constants (58-817 nM), kinetics of binding (5-60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI-TOF-MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP-SE MALDI-TOF-MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP-SE MALDI-TOF-MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes.

Screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis.

In response to the need for straightforward analytical methods to assess the affinity of molecularly imprinted nanoparticles (MIP NPs) for ligands, capillary electrophoresis (CE) was exploited using MIP NPs targeting the iron-regulating hormone hepcidin. In this work, MIP NPs were challenged with their template peptide, i.e., the N-terminal 5-mer of hepcidin, in comparison to unrelated ligand peptides. A CE separation method was developed ex novo achieving, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35 °C), the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed the estimation of a micromolar dissociation constant for the 5-mer template-MIP NPs complex, in agreement with independent measurements. The CE offered the advantages of a direct injection of the MIP NPs/ligand incubation mix, without preliminary fractionation steps, requiring only minimal sample volumes and short analysis times. In conclusion CE proved to be a valid technique for characterizing the interactions of MIP NP libraries for selected target compounds.

Surface plasmon resonance based on molecularly imprinted nanoparticles for the picomolar detection of the iron regulating hormone Hepcidin-25.

BACKGROUND: Molecularly imprinted polymer (MIP) technique is a powerful mean to produce tailor made synthetic recognition sites. Here precipitation polymerization was exploited to produce a library of MIP nanoparticles (NPs) targeting the N terminus of the hormone Hepcidin-25, whose serum levels correlate with iron dis-metabolisms and doping. Biotinylated MIP NPs were immobilized to NeutrAvidin™ SPR sensor chip. The response of the MIP NP sensor to Hepcidin-25 was studied. FINDINGS: Morphological analysis showed MIP NPs of 20-50 nm; MIP NP exhibited high affinity and selectivity for the target analyte: low nanomolar Kds for the interaction NP/Hepcidin-25, but none for the NP/non regulative Hepcidin-20. The MIP NP were integrated as recognition element in SPR allowing the detection of Hepcidin-25 in 3 min. Linearity was observed with the logarithm of Hepcidin-25 concentration in the range 7.2-720 pM. LOD was 5 pM. The response for Hepcidin-20 was limited. Hepcidin-25 determination in real serum samples spiked with known analyte concentrations was also attempted. CONCLUSION: The integration of MIP NP to SPR allowed the determination of Hepcidin-25 at picomolar concentrations in short times outperforming the actual state of art. Optimization is still needed for real sample measurements in view of future clinical applications.

Toward Nano- and Microplastic Sensors: Identification of Nano- and Microplastic Particles via Artificial Intelligence Combined with a Plasmonic Probe Functionalized with an Estrogen Receptor.

Nano- and microplastic particles are a global and emerging environmental issue that might pose potential threats to human health. The present work exploits artificial intelligence (AI) to identify nano- and microplastics in water by monitoring the interaction of the sample with a sensitive surface. An estrogen receptor (ER) grafted onto a gold surface, realized on a nonexpensive and easy-to-produce plastic optical fiber (POF) platform in order to excite a surface plasmon resonance (SPR) phenomenon, has been developed in order to carry out a "smart" sensitive interface (ER-SPR-POF interface). The ER-SPR-POF interface offers output data useful for exploiting a machine learning-based approach to achieve nano- and microplastic particle sensors. This work developed a proof-of-concept sensor through a training phase carried out by different particles, in terms of materials and size. The experimental results have demonstrated that the proposed "smart" ER-SPR-POF interface combined with AI can be used to identify the kind of particles in terms of the materials (polystyrene; poly(methyl methacrylate)) and size (20 µm; 100 nm) with an accuracy of 90.3%.

Porous Thermoplastic Molded Regenerated Silk Crosslinked by the Addition of Citric Acid.

Thermoplastic molded regenerated silk fibroin was proposed as a structural material in tissue engineering applications, mainly for application in bone. The protocol allows us to obtain a compact non-porous material with a compression modulus in the order of a Giga Pascal in dry conditions (and in the order of tens of MPa in wet conditions). This material is produced by compressing a lyophilized silk fibroin powder or sponge into a mold temperature higher than the glass transition temperature. The main purpose of the produced resin was the osteofixation and other structural applications in which the lack of porosity was not an issue. In this work, we introduced the use of citric acid in the thermoplastic molding protocol of silk fibroin to obtain porosity inside the structural material. The citric acid powder during the compression acted as a template for the pore formation. The mean pore diameter achieved by the addition of the higher amount of citric acid was around 5 µm. In addition, citric acid could effectively crosslink the silk fibroin chain, improving its mechanical strength. This effect was proved both by evaluating the compression modulus (the highest value recorded was 77 MPa in wet conditions) and by studying the spectra obtained by Fourier transform infrared spectroscopy. This protocol may be applied in the near future to the production of structural bone scaffolds.

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.

Estradiol Detection for Aquaculture Exploiting Plasmonic Spoon-Shaped Biosensors.

In this work, a surface plasmon resonance (SPR) biosensor based on a spoon-shaped waveguide combined with an estrogen receptor (ERα) was developed and characterized for the detection and the quantification of estradiol in real water samples. The fabrication process for realizing the SPR platform required a single step consisting of metal deposition on the surface of a polystyrene spoon-shaped waveguide featuring a built-in measuring cell. The biosensor was achieved by functionalizing the bowl sensitive surface with a specific estrogen receptor (ERα) that was able to bind the estradiol. In a first phase, the biosensor tests were performed in a phosphate buffer solution obtaining a limit of detection (LOD) equal to 0.1 pM. Then, in order to evaluate the biosensor's response in different real matrices related to aquaculture, its performances were examined in seawater and freshwater. The experimental results support the possibility of using the ERα-based biosensor for the screening of estradiol in both matrices.

Sensing Approaches Exploiting Molecularly Imprinted Nanoparticles and Lossy Mode Resonance in Polymer Optical Fibers.

In this work, two different lossy mode resonance (LMR) platforms based on plastic optical fibers (POFs) are developed and tested in a biochemical sensing scenario. The LMR platforms are based on the combination of two metal oxides (MOs), i.e., zirconium oxide (ZrO2) and titanium oxide (TiO2), and deposited on the exposed core of D-shaped POF chips. More specifically, two experimental sensor configurations were obtained by swapping the mutual position of the Mos films over to the core of the D-shaped POF probe. The POF-LMR sensors were first characterized as refractometers, proving the bulk sensitivities. Then, both the POF-LMR platforms were functionalized using molecularly imprinted nanoparticles (nanoMIPs) specific for human transferrin (HTR) in order to carry out binding tests. The achieved results report a bulk sensitivity equal to about 148 nm/RIU in the best sensor configuration, namely the POF-TiO2-ZrO2. In contrast, both optical configurations combined with nanoMIPs showed an ultra-low detection limit (fM), demonstrating excellent efficiency of the used receptor (nanoMIPs) and paving the way to disposable POF-LMR biochemical sensors that are easy-to-use, low-cost, and highly sensitive.

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.

Monocyte/macrophage proteomics: recent findings and biomedical applications.

Macrophages, originating from the migration and differentiation of circulating monocytes into virtually all tissues, are extremely flexible and plastic cells that play vital homeostatic roles, but also contribute to the pathophysiology of many human diseases. For these reasons, they are intensively studied by different approaches, recently including proteomics. Macrophage cells can be taken from a range of different sources, including blood monocytes and macrophages from tissues. Macrophages can also be generated by in vitro culture from blood monocytes, and cell lines derived from this lineage can be used. Similarly, many different proteomic techniques can be used, ranging from classic approaches based on 2D gel electrophoresis to more recent high-throughput gel-free techniques essentially based on mass spectrometry. Here, we review the application of such techniques to the study of monocytes/macrophages, and summarize some results potentially relevant to two paradigmatic conditions - atherosclerosis and disorders of iron metabolism.

Spoon-shaped polymer waveguides to excite multiple plasmonic phenomena: A multisensor based on antibody and molecularly imprinted nanoparticles to detect albumin concentrations over eight orders of magnitude.

A polymeric multimode waveguide, characterized by a pioneering spoon-shaped geometry, was herein proposed for the first time to devise Surface Plasmon Resonance (SPR) biochemical sensors. The plasmon excitation was enabled by layering a gold nanofilm of ∼60 nm onto the spoon-waveguide. As a consequence of the waveguide's extra-ordinary geometry, two distinct sensing regions were identified: a planar one, located on the spoon's neck, and a concave one on the bowl, with angled surfaces. The bulk sensitivity (Sn) is correlated both to the way the light was launched in/collected from the sensor (parallel or orthogonal to the main axis of the waveguide) and to the sensing area interrogated (planar-neck or angled-bowl), indicating that the sensor's performance can be conveniently tuned, depending on the chosen measuring configuration. The SPR sensor's characterization showed Sn equal to 750 nm/RIU for the neck and to 950 nm/RIU for the bowl. To further inspect the peculiar sensing-features and assess the application niches, the spoon-shaped waveguide was functionalized with two kinds of receptors, both specific for human serum albumin (HSA): an antibody on the bowl region (high Sn); molecularly imprinted nanoparticles (nanoMIPs) on the neck region (low Sn). The experimental results showed a limit of detection (LOD) for the immune-sensor of 280 pM and an LOD for the nanoMIP-sensor of 4.16 fM. The overall response of the HSA multi-sensor encompassed eight orders of magnitude, suggesting that the spoon-shaped waveguide's provides multi-scale detection and holds potential to devise multi-analyte sensing platforms.