Valentina Testa

Lithium is increasingly recognized as a strategic resource due to its essential role in lithium-ion batteries. The high demand for lithium is rapidly depleting these resources, making efficient recovery methods essential. Ion-imprinted polymers (IIPs) have emerged as a promising solution for this challenge. This study focuses on the innovative use of polyethylene glycol diacrylate (PEGDA) as both a crosslinker and functional monomer for synthesis of lithium-ion imprinted polymers (Li-IIP) based on the formation of pseudo-crown ether structures thanks to the template effect of the Li+ ions, providing an efficient and cheap alternative to the use of expensive crown ethers. The optimal Li-IIP formulation was identified with a PEGDA-575 to lithium molar ratio of 2:1, prepared in DMSO, with an equilibrium binding constant of 20.1 ± 4.5 x 103 mol L-1, an imprinting factor of 33.5, and a good selectivity towards Li+ ions respect to Na+, K+, Co2+, Ni2+ and Mn2+ ions of 0.17, 0, 0.42, 0.39 and 0.17, respectively. Additionally, the use of sodium carbonate as a leaching agent enhanced the binding capacity of the polymer to 0.18 mmol g-1 (1.25 mg g-1) offering a convenient alternative to traditional acidic leaching methods. Overall, these findings highlight the promising potential of PEGDA-based Li-IIPs to develop cheap and sustainable lithium recovery processes

Adenosine is a low molecular weight purine nucleoside whose immunoassay development is complicated by limited immunogenicity and by the close structural similarity among nucleosides and nucleotides. Here, we report a microplate-compatible competitive biomimetic immunoassay in which adenosine-imprinted nanoMIPs replace antibodies as the molecular recognition element. Four template-immobilization strategies were compared to define how template orientation affects imprinting efficiency. Direct immobilization through selective 2′,3′-ribose oxidation, which leaves the adenine base exposed during polymerization, produced the most effective binding sites; incorporation of an adenine-complementary 1-(vinylbenzyl)thymine monomer further increased the apparent equilibrium binding constant to 3.2 × 10⁵ L mol⁻¹. The selected nanoMIPs were implemented in a two-step competitive format in which free adenosine competes with a 5′-biotinylated polyadenine oligonucleotide for the imprinted sites, whereas signal generation is performed separately through streptavidin-horseradish peroxidase. Under optimized conditions, the assay provided a limit of detection of 2.3 ng mL⁻¹, a working range of 5-88 ng mL⁻¹, and an IC50 of 21.6 ± 0.05 ng mL⁻¹. Cross-reactivity (CR) experiments showed preferential recognition of adenine-containing compounds (CR values for ATP, AMP and guanosine were in the 20-48% range) whereas cytidine and thymidine did not produce measurable competition. By decoupling molecular competition from enzymatic signal generation, this work establishes a two-step nanoMIP assay architecture for adenosine and provides a transferable framework for translating synthetic receptors into antibody-free assays for challenging small-molecule targets.

Lithium is increasingly recognized as a strategic resource due to its essential role in lithium-ion batteries. The high demand for lithium is rapidly depleting these resources, making efficient recovery methods essential. Ion-imprinted polymers (IIPs) have emerged as a promising solution for this challenge. This study focuses on the innovative use of polyethylene glycol diacrylate (PEGDA) as both a crosslinker and functional monomer for synthesis of lithium-ion imprinted polymers (Li-IIP) based on the formation of pseudo-crown ether structures thanks to the template effect of the Li+ ions, providing an efficient and cheap alternative to the use of expensive crown ethers. The optimal Li-IIP formulation was identified with a PEGDA-575 to lithium molar ratio of 2:1, prepared in DMSO, with an equilibrium binding constant of 20.1 ± 4.5 x 10^3 mol L-1, an imprinting factor of 33.5, and a good selectivity towards Li+ ions respect to Na+, K+, Co2+, Ni2+ and Mn2+ ions of 0.17, 0, 0.42, 0.39 and 0.17, respectively. Additionally, the use of sodium carbonate as a leaching agent enhanced the binding capacity of the polymer to 0.18 mmol g-1 (1.25 mg g-1) offering a convenient alternative to traditional acidic leaching methods. Overall, these findings highlight the promising potential of PEGDA-based Li-IIPs to develop cheap and sustainable lithium recovery processes

The rapid detection of zinc in different aqueous matrices is very relevant. For example, a Zn²⁺ level above ca. 50 µM affects drinking water quality, while levels below ca. 25 µM in urine are related to higher probability of prostate cancer. Herein, a simple and rapid qualitative colorimetric sensor for the detection of zinc ions in aqueous samples is developed. The sensor exploits the reaction between 1,5-diphenylthiocarbazone and Zn²⁺ to form colored chelates whose color changes with increasing Zn²⁺ concentration. The chelating agent has been immobilized in a dried form on various cellulose- and synthetic-based materials to obtain a sensor that can be used for in situ analysis. The procedure to obtain the colorimetric device is easy and straightforward. Moreover, it requires neither specialized personnel to perform the analysis nor specialized personnel for the interpretation of the analytical results. The analysis requires only 20 µL of sample, and a reliable colorimetric output is obtained within 10 min and is stable up to 30 min. The sensor allows Zn²⁺ visual detection in drinking water and urine without any sample pre-treatment with excellent efficiency and repeatability. Considering the ability to distinguish between Zn²⁺ concentrations equal to 0.5 and 2× the cut-off level, the sensor showed sensitivity and specificity of 100% for fortified tap water analysis and 100% sensitivity and 88.9% specificity for urine samples. The almost-perfect concordance with the reference atomic absorption spectrometer and the 94.1% accuracy demonstrated the sensor’s excellent potential to be applied for selective qualitative Zn²⁺ detection in real-life situations.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic compound produced by certain fungi (e.g., Aspergillus flavus and Aspergillus parasiticus). Rapid and ultra-sensitive detection methods for AFB1 in various commodities are in high demand. This study aimed to enhance the sensitivity of a competitive lateral flow immunoassay (LFIA) for AFB1 detection by leveraging a previously developed experimental design strategy, named 4S. This approach comprises four phases—START, SHIFT, SHARPEN, and STOP—and involves the analysis of two reference conditions: NEG (0 ng/mL AFB1) and POS (1 ng/mL AFB1). By generating and overlaying response surfaces, regions of optimal NEG signal and POS/NEG signal ratio (IC%) were identified. Four variables were optimized: two related to the labeled antibody (its concentration and antibody-to-label ratio) and two to the competitor antigen (its concentration and hapten-to-protein ratio). An initial design defined the parameter space, while three subsequent designs did not yield further improvements in sensitivity. A strong anti-correlation was observed between the IC% and competitor parameters. The optimized LFIA-1 exhibited enhanced sensitivity, achieving a limit of detection of 0.027 ng/mL compared to 0.1 ng/mL for the original device. Additionally, the amount of expensive antibody required for device fabrication was reduced by around a factor of four.

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.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody–antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4–10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.

The solid-phase polymerization synthesis (SPPS) represents one of the most innovative approaches to the preparation of nano-sized molecularly imprinted polymers. One of its main features consists of the use of a solid support on which the template molecule is covalently grafted. It implies that the imprinting process does not involve the target molecule as is, but, rather, a structural modification of it. It is known that the rationally designed mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L)-phenylalanine (CHNA-Phe) is able to generate, by bulk polymerization, imprinted materials capable of recognizing the mycotoxin Ochratoxin A (OTA). In this work, we wanted to verify whether the CHNA-Phe can be a useful mimic template in the SPPS technique. The binding isotherm were measured in the pH range of 4–8 and the binding affinities for CHNA-Phe and OTA were compared, showing that CHNA-Phe-imprinted nanoMIPs recognize, in buffered water, equally well OTA, and that the overall molecular recognition depends markedly from pH-related ionic interactions between the ligand and the binding site. There results confirm that in the SPPS method, it is possible and convenient to use as mimic templates a molecule whose three-dimensional structure is to some extent different from the target without substantial loss of selectivity or binding affinity.

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.