Mass spectrometry (MS)-based structural proteomics can reveal higher-order structural features in complex biological samples, but many covalent footprinting and crosslinking strategies require specialized workflows to identify modified or crosslinked species. Here, we introduce a hydrogel-based strategy that converts protein surface accessibility into a simple signal-off readout in standard label-free LC-MS/MS. We synthesize protein-permeable polyethylene glycol molecularly imprinted hydrogels (MIHs) and install perfluorophenyl azide (PFPA) photoreactive groups after polymerization. When proteins are adsorbed to the imprinted cavities, UV irradiation crosslinks nearby protein surface segments to the gel. After extensive washing and in-gel digestion, peptides from crosslinked surface regions remain trapped in the gel matrix and are therefore depleted from the recovered digest, yielding a "missing-peptide" footprint without explicit identification of crosslinked species. Lysozyme-imprinted MIHs retain template recognition and report ligand-dependent protection of a carbohydrate-binding loop. A proteome-imprinted MIH generates > 3000 protein structural fingerprints from similar to 500 ng of HEK293T lysate and detects rapamycin-dependent changes in FK506-binding proteins (FKBPs), including an FKBP4 peptide near the rapamycin-binding interface. PFPA-functionalized MIHs thus provide an aqueous solid-phase transducer that makes structural proteomics compatible with routine bottom-up workflows.

The identification and targeting of lung-cancer-cell-surface proteins are important for drug development. Molecularly imprinted polymer nanoparticles (nanoMIPs) offer a synthetic approach for the recognition of proteins on the cell surfaces. This work outlines the use of a novel 'snapshot imprinting' approach to characterize differences in the cell-surface proteomes of lung cancer cell lines (A549, H460, H522) and a non-cancerous cell line (BEAS-2B) to potential protein targets for diagnostic and therapeutic applications. The mass spectrometry-based quantitative proteomics identified 2381 proteins. Fold change and p-value thresholds were used to define statistically and biologically significant differentially expressed proteins (DEPs) across cell lines, yielding 353, 426, and 274 DEPs for A549, H460, and H522, respectively, when compared to BEAS-2B. The DEPs identified across overlapping cell line comparisons were analyzed using Gene Ontology enrichment and a protein-protein network to identify hub proteins. Among these hub proteins, five proteins (NPM1, TOP2A, EZH2, PRKDC, and HNRNPK) were identified as clinically relevant when cross-referenced with the Human Protein Atlas database and the literature, highlighting their potential as diagnostic and therapeutic targets. These findings highlight the potential of nanoMIP-based snapshot imprinting as an alternative to 'classical' approaches for identifying potential protein targets for diagnostic and therapeutic applications.

The development of rapid, sensitive, and selective sensors for drug detection in biological fluids remains a critical challenge in clinical and forensic analysis. While localized surface plasmon resonance (LSPR) offers a powerful label-free detection mechanism, its selectivity in complex matrices typically relies on biological receptors that lack stability and are costly. To address this limitation, we developed a novel plasmonic sensing platform integrating gold nanoparticles (AuNPs) with molecularly imprinted polymers (MIPs) to create robust synthetic receptors termed AuNP@MIP nanosensors and demonstrated its application for amphetamine detection. The AuNP@MIP composites were synthesized via solid-phase polymerization, and the effect of AuNP core size (5-100 nm) on sensor performance was systematically optimized. Comprehensive characterization using dynamic light scattering (DLS) and transmission electron microscopy (TEM) confirmed the composite morphology and homogeneity. The optimized LSPR sensor exhibited exceptional sensitivity and selectivity for amphetamine, achieving detection limits of 0.24 nM in buffer, 0.36 nM in urine, and 0.50 nM in plasma, with a linear range of 0.3-1.0 nM across all matrices, confirmed by triplicate measurements. No response was observed for paracetamol, while selectivity against structural analogues (methamphetamine and phenethylamine) showed negligible cross-reactivity (<0.2% for methamphetamine and <0.4% for phenethylamine). The AuNP@MIP platform combines the high sensitivity of plasmonic transduction with the tailor-made selectivity and robustness of MIPs, enabling rapid, real-time analysis directly in complex matrices without extensive sample preparation. This work establishes a versatile and powerful strategy for on-site drug monitoring, paving the way for next-generation, reagent-free biosensors.

Enzymes are highly complex nanomachines produced by cells with the ability to catalyse diverse chemical reactions. Their applications in biotechnology and molecular diagnostics are widespread. However, most enzymes suffer from poor operational and storage stability and high manufacturing costs. Identifying suitable enzyme for particular substrates of practical importance is often challenging. These limitations are driving the search for synthetic alternatives - nanoparticles with catalytic properties that can mimic enzymatic reactions (nanozymes). A broad variety of organic and inorganic nanoparticles have been developed with catalytic power matching that of natural enzymes. Unfortunately, developing nanozymes with substrate/ligand specificity akin to that of enzymes and antibodies has proven challenging. Here we report a novel generic strategy for the synthesis of highly specific nanozymes mimicking peroxidase based on MIP nanoparticles with gold cores, prepared by a Fenton-like reaction. Synthesis of MIP shells was achieved by localised radical polymerization of monomer mixture triggered by hydroxyl radicals produced by hydrogen peroxide decomposed on the gold surface. The products of this reaction are highly specific and robust composite Au/MIP nanoparticles (Au/MIP nanozymes) with integrated biorecognition and catalytic properties. In this work we also explore the use of synthesised Au/MIP nanozymes in oxidation of BPR by hydrogen peroxide for colorimetric detection of template analytes such as amphetamine. The developed assay allowed the detection of corresponding analytes at nanomolar concentrations in complex biological matrices. The assay was robust and easy to perform with minimal operational steps. These findings hold great promise for developing a new form of homogeneous, completely abiotic, highly sensitive, highly specific user-friendly assays for in vitro diagnostics.

A class of hybrid molecularly imprinted polymeric nanoparticles (nanoMIPs) comprising the in situ formation of gold nanoparticles (AuNPs) immobilised in a molecularly imprinted D-gluconate polymer has been designed with the objective of attempting the electrochemical quantification of gluconic acid (GA) in a wine setting. The imprinted polymers were synthesised in the presence of AuNP precursors in a pre-polymerisation mixture, which were confined to one another during the polymerisation of the chains. This allowed the formation of hybrid electroactive responsive imprinted nanoparticles (hybrid AuNPs@GA-nanoMIP), which exhibited enhanced electron conductivity. The morphological characterisation of the produced nanoMIPs revealed a fully decorated Au spherical surface of 200 nm in diameter. This resulted in a large active surface area distribution, as well a pronounced electrochemical peak response at the commercial screen-printed platinum electrode (SPPtE), accompanied by enhanced electron kinetics. The AuNPs@GA-nanoMIP sensor demonstrated the ability to detect a broad range of GA concentrations (0.025–5 mg/mL) with exceptional selectivity and reproducibility. The calibration curves were fitted with different isotherm models, such as the Langmuir, Freundlich and Langmuir–Freundlich functions. Moreover, the efficacy of the detection method was demonstrated by the recovery rates observed in real samples of Italian red wine. This research contributes to the development of a robust and reliable electrochemical sensor for the on-site determination of gluconic acid in food analysis.

BACKGROUND:Biogenic amines (BAs) are low molecular weight nitrogenous compounds present in various foods. While physiologically essential in trace amounts, elevated concentrations of BAs pose health risks, including serving as precursors to carcinogens. In fermented foods such as kumiss-a traditional fermented horse milk-the quantitative assessment of BAs is critical due to their health implications. However, the wide range of logP and pKa values among BAs presents significant challenges for their simultaneous extraction and accurate analysis. RESULT:This study introduces a novel approach for the simultaneous extraction of eight BAs in kumiss using dummy molecularly imprinted polymer (DMIP) coated fibers. The DMIP was synthesized using diethylamine dansyl chloride as a derivatized template, enabling selective recognition of eight BA derivatives. The fabricated coated fibers, which are reusable and cost-effective, were integrated into an array device for high-throughput solid-phase microextraction (SPME), achieving an average extraction time of less than 2 min per sample. The SPME method demonstrated high recoveries (70.06-110.92 %) when coupled with high-performance liquid chromatography (HPLC) analysis. Linear calibration curves were established between the peak area and the concentration of BAs over the range of 0.2-10 mg L-1, with high regression coefficients (>0.99) and low detection limits (0.025-0.123 mg L-1). The DMIP coated fiber array extraction device provided highly selective and efficient separation of BA derivatives from complex matrices, as successfully demonstrated using kumiss samples. SIGNIFICANCE AND NOVELTY:This study presents a novel dummy molecular imprinting strategy for the fabrication of DMIP coated fiber array for SPME, addressing the limitations of traditional methods in the simultaneous recognition of structurally diverse BAs. This approach significantly enhances the efficiency and selectivity of BA analysis, which is essential for the quality control of fermented foods.

Molecularly imprinted polymers are highly valued in diverse fields due to their overall stability, molecular recognition capabilities, and low-cost of preparation. Solid-phase synthesis has been an important imprinting strategy developed in the past decade. Distinct from conventional imprinting methods, the solid-phase synthesis approach immobilizes the template molecules onto the substrate. The immobilization of template molecules not only avoids the leakage of template molecules but also enables the reuse of template molecules and improving specificity of polymer binding sites. During preparation, low-affinity molecularly imprinted polymer nanoparticles (nanoMIPs) can be separated while high affinity nanoMIPs are subsequently collected. Remarkably, nanoMIPs have demonstrated comparable or even superior performance to biological antibodies in diverse biomedical and trace detection applications. This comprehensive review covers the whole process of preparation, characterization, and application of nanoMIPs synthesized through the solid-phase synthesis. Furthermore, it provides an analysis of the status of the technique and a discussion on future development.

This study introduces a novel one-step magnetic immunoassay (mMINA) designed for effective and sensitive detection of fentanyl, a potent synthetic opioid. The proposed method offers several advantages over the standard ELISA protocol, notably a substantial reduction in procedural steps from sample application to analysis. Simplicity directly translates into saved time, lowered measurement costs, minimized potential for errors, and enhanced user-friendliness and precision. The assay utilizes 96-well magnetic microplates for solid-phase separation, fluorescently labeled anti-fentanyl antibodies, and fentanyl-functionalized iron oxide nanoparticles as competing probes. Upon co-incubation, free fentanyl in the sample competes with nanoparticle-bound fentanyl for limited antibody binding, resulting in an inversely proportional fluorescence signal. The method demonstrated high specificity and sensitivity, with a limit of detection (LoD) of 1.562 nM in blood plasma, and showed high selectivity with structurally similar opioids such as morphine, heroin, or cocaine. It maintained performance in complex biological matrices, underscoring its robustness and reproducibility. Importantly, the modular platform design, based on commercially available antibodies, allows adaptation to other small-molecule targets. This flexibility, combined with operational simplicity and low cost, positions the mMINA assay as a practical tool for rapid drug screening in clinical, forensic, and field-based settings.

In this study, a homogeneous fluorescence approach for detecting tryptamine (TRY) was developed by integrating fluorescent molecularly imprinted polymer nanoparticles (F-nanoMIPs) with Förster resonance energy transfer (FRET). The F-nanoMIPs were synthesized via solid-phase imprinting using TRY as a template and 2-(5-(dimethylamino)naphthalen-1-ylsulfonyloxy)ethyl methacrylate (DNS-HEMA) as a fluorescent monomer. Binding properties were evaluated through isothermal adsorption experiments, which revealed that F-nanoMIPs exhibited a high adsorption capacity (289.06 mg·g-1) for TRY. The FRET-based detection system demonstrated rapid detection within 1 min, with a linear range of 0.678-67.842 μM (R2 = 0.9946) and a low limit of detection of 0.554 μM. Cross-reactivity studies highlighted remarkable selectivity for TRY and its analogues (< 3 %). In spiked fruit samples, the recoveries for TRY ranged from 85.58 to 110.25 %. This approach combines molecular imprinting technology with FRET mechanisms to establish a cost-effective detection sensor featuring long-term stability, rapid response, and dual recognition capabilities.

A new voltammetric sensor was developed for the sensitive and selective detection of perfluorooctanoic acid (PFOA) in water. The sensor is based on molecularly imprinted polymer nanoparticles (nanoMIPs) labelled with a redox-active moiety, which imparts electrochemical properties to the polymer. These nanoMIPs were synthesized using solid-phase polymerization, with their size optimized through chemometric modelling, including functional monomer screening and recognition cavity design. The optimization aimed to minimize the nanoMIP size, which is directly influenced by the amount of polymerization initiator, and to enhance sensor response. A commercially available screen-printed platinum electrode (SPPtE) served as the transducer element, functionalized with APTES to enable covalent attachment of nanoMIPs. The sensor demonstrated a detection capability for PFOA concentrations as low as 0.40 f 0.03 pg mL- 1, and a linear response up to 7.5 pg mL- 1. Furthermore, the calibration curve was fitted to the Michaelis-Menten model, thanks to the allosteric behavior of the nanoparticles mimicking enzyme-substrate interactions. A Km of 4.34 pg mL- 1 for MIP and 17.73 pg mL- 1 for the control were obtained. Its selectivity was evaluated in samples containing structurally similar PFAS compounds, showing high effectiveness also in contaminated water. Additionally, a successful regeneration strategy was developed, enabling the sensor to be used across at least 3 work cycles. The successful adaptation of the developed method to a handheld miniaturized potentiostat connected to an Android phone enables on-site inspections of surface waters at the point of need.

A handheld smartphone-compatible molecularly imprinted polymer (MIP)-based sensor was developed for the analysis of bisphenol A (BPA) in wastewater samples. Sensing elements based on ethylene glycol methacrylate phosphate (EGMP)-containing MIP films were designed and optimized using molecular dynamics simulations. The highly porous MIP films were synthesized via in situ polymerization, employing a fragment-based approach. The colorimetric response was based on the 4-aminoantipyrine method, while the MIP films were further utilized to detect BPA with a smartphone. The proposed sensor exhibited a wide linear range from 5 to 250 μM, with a limit of detection (LOD) of 5 μM (S/N = 3). Furthermore, the designed analytical system demonstrated excellent analytical performance in terms of selectivity, stability, and reproducibility. During sensor validation, real wastewater samples were successfully tested for BPA, showcasing the feasibility of the smartphone-compatible MIP-based sensor. Recovery values of 87.1–114.6

Correction for 'An impedimetric sensor based on molecularly imprinted nanoparticles for the determination of trypsin in artificial matrices - towards point-of-care diagnostics' by Sabrina Di Masi et al., Anal. Methods, 2024, 16, 742-750, https://doi.org/10.1039/D3AY01762A.

A portable and highly sensitive sensor was designed for the specific detection of 3,4-methyl-enedioxy-methamphetamine (MDMA), in a range of field-testing situations. The sensor can detect MDMA in street samples, even when other controlled substances drugs, or adulterants are present. In this work, we report for the first time a sensor using electroactive molecularly imprinted polymer nanoparticles computationally designed to recognize MDMA and then produced using solid phase synthesis. A composite comprising chitosan, reduced graphene oxide, and molecularly imprinted polymer nanoparticles synthesized for MDMA for the first time was immobilized on screen-printed carbon electrodes. The sensors displayed a satisfactory sensitivity (106.8 nA × μM-1), limit of detection (1.6 nM; 0.31 ng/mL), and recoveries (92-99%). The accuracy of the results was confirmed through validation using Ultra-High Performance Liquid Chromatography coupled with tandem Mass Spectrometry (UPLC-MS/MS). This technology could be used in forensic analysis and make it possible to selectively detect MDMA in street samples.

Adeno-associated virus (AAV)-derived viral vectors are a promising platform for the delivery of curative, life-changing therapies to a huge number of patients with monogenic disorders. There are currently over 250 clinical trials ongoing worldwide. However, for these therapies to benefit as many patients as possible, techniques must be developed to treat those with pre-existing immunity and to potentially allow re-administration of a dose in the future, should efficacy wane over time. This review discusses the current state and prospects of technologies to evade and overcome these immune responses and allow successful treatment of the greatest number of patients possible.

Redox-active molecularly imprinted polymer nanoparticles selective for glyphosate, MIP-Gly NPs, were devised, synthesized, and subsequently integrated onto platinum screen-printed electrodes (Pt-SPEs) to fabricate a chemosensor for selective determination of glyphosate (Gly) without the need for redox probe in the test solution. That was because, ferrocenylmethyl methacrylate was added to the polymerization mixtures during the NPs synthesis so that the resulting MIP-Gly NPs contained covalently immobilized ferrocenyl moieties as the reporting redox ingredient, conferring these NPs with electroactive properties. MIP-Gly NPs of four different compositions were evaluated. The herein described approach represents a simple and effective way to endow MIP NPs with electrochemical reporting capabilities with neither the need to functionalize them post-synthesis nor to use electrochemical mediators present in the tested solution during the analyte determinations. MIP-Gly NPs synthesized using allylamine and squaramide-based monomers appeared most selective to Gly. The Pt-SPEs modified with MIP-Gly NPs were characterized with differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Changes in the DPV peak originating from the oxidation of the ferrocenyl moieties in these MIP-Gly NPs served as the analytical signal. The DPV limit of detection and the linear dynamic concentration range for Gly were 3.7 pM and 25 pM-500 pM, respectively. Moreover, the selectivity of the fabricated chemosensors was sufficiently high to determine Gly successfully in spiked river water samples.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

Cadmium is a highly toxic heavy metal, even at low concentrations. Moreover, it bio-accumulates with a long biological half-life producing a wide variety of acute and chronic effects like cancer in humans. Therefore, there is significant interest in providing a portable and user-friendly sensor for heavy metal detection. A highly sensitive and selective electrochemical sensor for Cd(II) determination was developed as an alternative. The Cd (II) receptors were integrated into the sensor using an ion-imprinted polymer film (IIPs film). Accordingly, 4-aminophenylacetic acid (4-APA) was electropolymerised in the presence of Cd(II) ions as a template using cyclic voltammetry (CV) on screen-printed carbon electrodes (SPCEs). Imprinted cavities were obtained after the alkaline elution of ions. Sensor characterisation was performed using SEM, ATR-FTIR and electrochemical methods, such as cyclic voltammetry (CV) and electrochemical Impedance Spectroscopy (EIS), and compared to control experiments (not imprinted polymeric film, NIP film). Analytical performances of the IIP sensor revealed a sensitivity 5-order of magnitude higher than the NIP response in a concentration range from 10 to 1200 nM of Cd (II). This technology can potentially be applied for water quality control and monitoring of heavy metals.

The paper is a self-review of works on development of new approaches to formation of mimics of receptor and catalytic sites of biological macromolecules in the structure of highly cross-linked polymer membranes and thin films. The general strategy for formation of the binding sites in molecularly imprinted polymer (MIP) membranes and thin films was described. A selective recognition of a number of food toxins, endocrine disruptors and metabolites is based on the results of computational modeling data for the prediction and optimization of their structure. A strategy proposed for the design of the artificial binding sites in MIP membranes was supported by the research performed by the authors on development of a number of the MIP membrane-based affinity and catalytic biosensors for selective and sensitive measurement (detection limits 0.3-100 nM) of the target analytes. Novel versatile approaches aimed at improving sensitivity of the developed biosensor systems were discussed.

novel molecular imprinting technique is used to map and identify four antigen-bindingfragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodiescapable of binding to AAV. The presented proof-of-principle study demonstrates thatsynthesised Fab peptides, identified by molecular imprinting, can prevent AAV8binding to antibodies. A study shows the opportunity to use Fab peptides specific toAAV as a formulation for improving the efficiency of gene therapy by preventing theimmune response.