Lumpy skin disease (LSD) is an infectious disease affecting bovine with severe symptomatology. The implementation of effective control strategies to prevent infection outbreak requires rapid diagnostic tools. Two monoclonal antibodies (mAbs), targeting different epitopes of the LSDV structural protein p32, and gold nanoparticles (AuNPs) were used to set up a colorimetric sandwich-type lateral flow immunoassay (LFIA). Combinations including one or two mAbs, used either as the capture or detection reagent, were explored to investigate the hook effect due to antigen saturation by the detector antibody. The mAb-AuNP preparations were optimized by a full-factorial design of experiment to achieve maximum sensitivity. Opposite optimal conditions were selected when one Mab was used for capture and detection instead of two mAbs; thus, two rational routes for developing a highly sensitive LFIA according to Mab availability were outlined. The optimal LFIA for LSDV showed a low limit of detection (10^3.4 TCID₅₀/mL), high inter- and intra-assay repeatability (CV% < 5.3%), and specificity (no cross-reaction towards 12 other viruses was observed), thus proving to be a good candidate as a useful tool for the point-of-need diagnosis of LSD.

Solid phase synthesis (SPS) of molecularly imprinted nanopolymers (nanoMIPs) represents an innovative method to prepare nanomaterials with tailor-made molecular recognition properties towards peptides and proteins. The synthesis of nanoMIPs by SPS usually involves a pre-polymerization formulation, where the cross-linker is invariably N,N′-methylen-bis-acrylamide (BIS). To date, the effect of cross-linkers on the binding properties of nanoMIPs prepared using cross-linkers other than BIS has never been reported. In this work, in order to investigate the effect of different cross-linkers in protein-imprinted nanoMIPs prepared by SPS, alongside BIS we considered other similar cross-linkers: N,N′-ethylene dimethacrylamide (EDAM), N,O-bis-methacryloylethanolamine (NOBE), ethylene glycol dimethacrilate (EDMA) and glycerol dimethacrylate (GDMA), replacing BIS with them in pre-polymerization mixtures. The synthetized nanoMIPs were homogeneous, with a polydispersity index of 0.24–0.30 and a mean diameter of 129–169 nm in water. The binding properties of the nanoMIPs were measured via equilibrium partition experiments with the template, rabbit IgG (RIgG), and the selectivity was evaluated with respect to bovine IgG (BIgG), bovine serum albumin (BSA) and hen egg lysozyme (LZM). The experimental results show that all the cross-linkers, with the exception of EDMA, endowed nanoMIPs with high binding affinities for the template (BIS: 16.0 × 10⁶ mol⁻¹ L, EDAM: 8.8 × 10⁶ mol⁻¹ L, NOBE: 15.8 × 10⁶ mol⁻¹ L, and GDMA: 12.8 × 10⁶ mol⁻¹ L), medium to high imprinting factors (BIS: 12.3, EDAM: 5.5, NOBE: 7.2, and GDMA: 11.6) and good selectivity towards other proteins but markedly dependent on the structure of the cross-linker, confirming the importance of the latter in the SPS of imprinted nanopolymers.

NanoMIPs that are prepared by solid phase synthesis have proven to be very versatile, but to date only limited attention has been paid to their use in solid phase extraction. Thus, since nanoMIPs show close similarities, in terms of binding behavior, to antibodies, it seems relevant to verify if it is possible to use them as mimics of the natural antibodies that are used in immunoextraction methods. As a proof-of-concept, we considered prepared nanoMIPs against fluoroquinolone ciprofloxacin. Several nanoMIPs were prepared in water with polymerization mixtures of different compositions. The polymer with the highest affinity towards ciprofloxacin was then grafted onto a solid support and used to set up a solid phase extraction–HPLC method with fluorescence detection, for the determination of fluoroquinolones in human urine. The method resulted in successful selection for the fluoroquinolone antibiotics, such that the nanoMIPs were suitable for direct extraction of the antibiotics from the urine samples at the µg mL⁻¹ level. They required no preliminary treatment, except for a 1 + 9 (v/v) dilution with a buffer of pH 4.5 and they had good analyte recovery rates; up to 85% with precision in the range of 3 to 4.5%, without interference from the matrix. These experimental results demonstrate, for the first time, the feasibility of the use of nanoMIPs to develop solid phase extraction methods.

The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0–05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1–18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32–50 mol%), characterized by non-specific nanopolymer–ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10–20 mol%, well higher than those normally used in this technique.