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

Phosphoproteomics

The book provides an overview of state-of-art techniques for the purification, analysis and quantification of proteins in complex samples using different enrichment strategies

RULES TO PREPARE PEPTIDE-IMPRINTED NANOGELS WITH HIGH-AFFINITY BINDING SUITABLE FOR SENSING AND ASSAYS BY PRECIPITATION POLYMERIZATION

Molecular imprinting is a technique for preparing polymeric scaffolds (Molecularly Imprinted Polymers, MIPs) that function as synthetic receptors and show affinity and selectivity towards a target analyte. The most attractive characteristic of MIPs is the possibility to tailor the binding selectivity so to gain recognition levels on the par of biological receptors. When MIPs are downsized to the nanoscale (nanoMIPs), they show an increase in the number of accessible imprinted binding cavities per material weight and an enhanced molecular recognition ability, leading to faster binding kinetics, higher affinity and selectivity, thus strengthening the resemblance to antibodies and natural receptors. Being the recognition properties of the nanoMIPs strictly correlated to the effective formation of the imprints in the chosen synthetic conditions, a deeper comprehension of the polymerization at the nanoscale is required. In order to fill this lack, we studied the best conditions to form imprints at the nanoscale when the synthesis occurs by a precipitation polymerization protocol by means of an one-pot synthesis via free radical initiation in aqueous solution, using as target analyte the peptide of Troponin I, clinical marker of cardiac failure. By exploring a range of monomers combinations, polyacrylamide-based MIP nanogels having homogeneous nano-dimensions and a low number of binding sites per nanoparticle were synthesized. To this purpose, we evaluated the influence of the monomer composition and the total monomers to template molar ratio on the hydrodynamic sizes and on the recognition properties, respectively, defining the conditions to tune the nanoMIP dimensions (from 60 to >600 nm) and to improve the efficacy of the imprinting process. In the light of the achieved results, the present work contributes to define the best conditions to obtain imprinted peptides at the nanoscale and impact on the production of synthetic recognition materials suitable for sensing and assays

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

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

Substitution of antibodies and receptors with molecularly imprinted polymers in enzyme-linked and fluorescent assays

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid, 3- thiopheneboronic acid and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in ELISA

Molecularly Imprinted Polymers for Cell Recognition

Since their conception 50 years ago, molecularly imprinted polymers (MIPs) have seen extensive development both in terms of synthetic routes and applications. Cells are perhaps the most challenging target for molecular imprinting. Although early work was based almost entirely around microprinting methods, recent developments have shifted towards epitope imprinting to generate MIP nanoparticles (NPs). Simultaneously, the development of techniques such as solid phase MIP synthesis has solved many historic issues of MIP production. This review briefly describes various approaches used in cell imprinting with a focus on applications of the created materials in imaging, drug delivery, diagnostics, and tissue engineering

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

A plasmonic gold nano-surface functionalized with the estrogen receptor for fast and highly sensitive detection of nanoplastics

: Nanoplastics are a global emerging environmental problem whose effects might pose potential threats to the human's health. Despite the relevance of the issue, fast, reliable and quantitative in situ analytical approaches to determine nanoplastics are not yet available. The aim of this work was to devise an optical sensor with the goal of direct detecting and quantifying nanoplastics in seawater without sample pre-treatments. To this purpose, a nano-plasmonic biosensor was developed by exploiting an Estrogen Receptor (ER) recognition element grafted onto a polymer-based gold nanograting (GNG) plasmonic platform. The ER-GNG biosensor required just minute sample volumes (2 μL), allowed rapid detection (3 min) and enabled to determine nanoplastics in simulated seawater with a linear dynamic concentrations range of 1-100 ng/mL, thus encompassing the expected environmental loads. The nanostructured grating (GNG) provided remarkable performance enhancements, extending the measurement range across five orders of magnitude, thanks to the both the SPR and the localized SPR phenomena occurring at the GNG chip. At last, the ER-GNG biosensor was tested on real seawater samples collected in the Naples area and the results (∼30 ng/mL) were verified by a conventional approach (filtration and evaporation), confirming the ER-GNG sensor offers a straightforward and highly sensitive method for the direct in-field nanoplastics monitoring

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