Francesco Canfarotta

A pseudo-ELISA based on molecularly imprinted nanoparticles for detection of gentamicin in real samples

The enzyme-linked immunosorbent assay (ELISA) is one of the most widely employed tests in diagnostics, and it relies on the use of antibodies to quantify the molecule of interest. Molecularly imprinted nanoparticles (nanoMIPs), thanks to their stability, cost efficiency and easy production, are a promising alternative to antibodies in assays and sensors. In this work, nanoMIPs have been produced by means of a solid-phase approach and employed for the detection of gentamicin in real samples. The produced nanoMIPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The determination of gentamicin in spiked milk was implemented through an assay similar to enzyme-linked immunosorbent assay, in which the nanoMIPs were used as a synthetic capture antibody (pseudo-ELISA). The detection of gentamicin was achieved in competitive binding experiments with a horseradish peroxidase–gentamicin conjugate. Gentamicin was determined in milk at clinically relevant concentrations with a mean accuracy of 94%. The cross-reactivity of such nanoparticles was investigated with streptomycin and ampicillin as control antibiotics, demonstrating excellent specificity.

Biocompatibility and internalization of molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIP) are receiving increasing attention thanks to their robustness, stability, and inexpensive manufacture compared with their bio-analogues such as antibodies. The molecular imprinting process can be defined as the generation of molecular recognition sites in a synthetic polymer. The template-derived sites created within a polymeric matrix allow MIPs (often referred as plastic antibodies) to selectively recognize and bind to the target molecule. Therefore, MIPs can be used in sensors and in separation and diagnostics. Owing to their size and functional properties, MIP nanoparticles (NPs) can potentially be used in biomedicine, but comprehensive analysis of their interaction with cells and in vitro toxicological tests must be performed first. Herein, we report the synthesis of bare and core–shell imprinted NPs using an innovative solid-phase approach and the toxicological evaluation of such NPs in different cell lines (HaCaT, MEFs, HT1080, and macrophages). We also evaluated the influence of the protein corona on particle stability, the internalization of NPs in cells, and the influence of various surface coatings. Studies on the metabolic effects of imprinted NPs on fibroblasts showed that bare MIPs do not alter cell metabolism, whereas some issues arise when specific particle coatings are used. Furthermore, in vitro cytokine release studies revealed that macrophages were not activated in the presence of the MIPs evaluated in this study. The results suggest that MIP NPs are biocompatible, paving the way for their in vivo application.

Replacement of Antibodies in Pseudo-ELISAs: Molecularly Imprinted Nanoparticles for Vancomycin Detection

The enzyme-linked immunosorbent assay (ELISA) is a widely employed analytical test used to quantify a given molecule. It relies on the use of specific antibodies, linked to an enzyme, to target the desired molecule. The reaction between the enzyme and its substrate gives rise to the analytical signal that can be quantified. Thanks to their robustness and low cost, molecularly imprinted polymer nanoparticles (nanoMIPs) are a viable alternative to antibodies. Herein, we describe the synthesis of nanoMIPs imprinted for vancomycin and their subsequent application in an ELISA-like format for direct replacement of antibodies.

A novel capacitive sensor based on molecularly imprinted nanoparticles as recognition elements

Molecularly Imprinted Polymers (MIPs) are synthetic receptors capable of selective binding to their target (template) molecules and, hence, are used as recognition elements in assays and sensors as a replacement for relatively unstable enzymes and antibodies. Herein, we describe a manufacturing-friendly protocol for integration of MIP nanoparticles (nanoMIPs) with a (label-free) capacitive sensor. The nanoMIPs were produced by solid-phase synthesis for two templates with different sizes and properties, including a small molecule tetrahydrocannabinol (THC) and a protein (trypsin). NanoMIPs were deposited on the surface of the sensor and the change in capacitance (ΔC) upon binding of the target was measured. The significant improvement in the selectivity and limit of detection (one order of magnitude compared to previously used MIP microparticles) can be attributed to their increased surface-to-volume ratio and higher specificity of the nanoMIPs produced by the solid-phase method. The methodology described is also compatible with common sensor fabrication approaches, as opposed to methods involving in situ MIP polymerisation. The proposed sensor shows high selectivity, fast sensor response (45 min including injection, regeneration and re-equilibration with running buffer), and straightforward data analysis, which makes it viable for label-free monitoring in real-time. The set of targets assessed in this manuscript shows the general applicability of the biosensor platform.

Biomimetic Silica Nanoparticles Prepared by a Combination of Solid-Phase Imprinting and Ostwald Ripening.

Herein we describe the preparation of molecularly imprinted silica nanoparticles by Ostwald ripening in the presence of molecular templates immobilised on glass beads (the solid-phase). To achieve this, a seed material (12 nm diameter silica nanoparticles) was incubated in phosphate buffer in the presence of the solid-phase. Phosphate ions act as a catalyst in the ripening process which is driven by differences in surface energy between particles of different size, leading to the preferential growth of larger particles. Material deposited in the vicinity of template molecules results in the formation of sol-gel molecular imprints after around 2 hours. Selective washing and elution allows the higher affinity nanoparticles to be isolated. Unlike other strategies commonly used to prepare imprinted silica nanoparticles this approach is extremely simple in nature and can be performed under physiological conditions, making it suitable for imprinting whole proteins and other biomacromolecules in their native conformations. We have demonstrated the generic nature of this method by preparing imprinted silica nanoparticles against targets of varying molecular mass (melamine, vancomycin and trypsin). Binding to the imprinted particles was demonstrated in an immunoassay (ELISA) format in buffer and complex media (milk or blood plasma) with sub-nM detection ability.

Specific Drug Delivery to Cancer Cells with Double-Imprinted Nanoparticles against Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is over-expressed in many tumors, including almost half of triple-negative breast cancers. The latter belong to a very-aggressive and drug-resistant form of malignancy. Although humanized anti-EGFR antibodies can work efficiently against these cancers both as monotherapy and in combination with genotoxic drugs, instability and high production costs are some of their known drawbacks in clinical use. In addition, the development of antibodies to target membrane proteins is a very challenging task. Accordingly, the main focus of the present work is the design of supramolecular agents for the targeting of membrane proteins in cancer cells and, hence, more-specific drug delivery. These were produced using a novel double-imprinting approach based on the solid-phase method for preparation of molecularly imprinted polymer nanoparticles (nanoMIPs), which were loaded with doxorubicin and targeted toward a linear epitope of EGFR. Additionally, upon binding, doxorubicin-loaded anti-EGFR nanoMIPs elicited cytotoxicity and apoptosis only in those cells that over-expressed EGFR. Thus, this approach can provide a plausible alternative to conventional antibodies and sets up a new paradigm for the therapeutic application of this class of materials against clinically relevant targets. Furthermore, nanoMIPs can promote the development of cell imaging tools against difficult targets such as membrane proteins.

Novel assay format for proteins based on magnetic molecularly imprinted polymer nanoparticles—detection of pepsin

A novel, abiotic assay format for the quantification of pepsin has been developed featuring replacement of conventional antibody binders and enzyme reporters with entirely synthetic alternatives. T...