Correction: An impedimetric sensor based on molecularly imprinted nanoparticles for the determination of trypsin in artificial matrices - towards point-of-care diagnostics.

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.

An impedimetric sensor based on molecularly imprinted nanoparticles for the determination of trypsin in artificial matrices - towards point-of-care diagnostics.

A high-performance impedimetric sensing platform was designed to detect proteins by employing molecularly imprinted polymeric nanoparticles (nanoMIPs) as selective receptors. This was achieved via the combination of the nanoMIPs with a self-assembled thioctic acid (SAM-TA) monolayer onto screen-printed gold electrodes, providing stable covalent attachment of the selective binder to the transducer. Taguchi design has been modelled to achieve the optimal level of sensor fabrication parameters and to maximise the immobilisation of nanoMIPs and their response (e.g. the response of imprinted polymers compared with the non-imprinted control). The developed sensor was tested towards a range of concentrations of trypsin dissolved in ammonium acetate (pH = 6) and showed promising applicability in artificial saliva, with a recovery percentage between 103 and 107%.

Design of smart nanoparticles for the electrochemical detection of 3,4-methylenedioxymethamphetamine to allow in field screening by law enforcement officers.

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.

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

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.

A Biomimetic Polymer for the Extraction and Purification of Superior Analogues of Amphotericin B.

Amphotericin B has been an essential drug in the fight against leishmaniasis and fungal pathogens for decades, and has more recently gained attention for the very limited microbial resistance displayed against it. However, its toxicity has restricted its use to only the most severe cases of disease, and attempts to reduce these ill effects via formulation have had only minor success. Genetic engineering has allowed the development of superior amphotericin analogues, notably 16-descarboxyl-16-methyl amphotericin B (MeAmB), which shows a ten-fold reduction in toxicity in addition to a slight improvement in therapeutic activity. However, MeAmB is difficult to extract from its bacterial source and purify. Presented here is an alternative method of MeAmB purification. A biomimetic polymer with a high affinity for MeAmB was designed via computational modelling and synthesised. Prepared as a separation column, the polymer was able to retain the target MeAmB whilst allowing the removal of cell debris from the bacterial extract. Starting with a simple bacterial extract, the relatively simple process allowed the purification of an MeAmB salt complex at approximately 70% MeAmB, and likely higher purification from further extraction. The mean MeAmB recovery between the pre-purification extract sample and the final product was 81%. This is the first successful demonstration of extraction or purification of any amphotericin molecule with any polymeric material. The biomimetic polymer was additionally reusable and simple to fabricate, giving this technique significant advantages over traditional methods of extraction and purification of valuable compounds.

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation.

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

Electroactive molecularly imprinted polymer nanoparticles for selective glyphosate determination.

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.

An enhanced fluorescent sensor system based on molecularly imprinted polymer chips with silver nanoparticles for highly-sensitive zearalenone analysis.

A novel enhanced fluorescent sensor system for zearalenone (ZON) determination in flour samples is presented. The ZON-selective molecularly imprinted polymer (MIP) films were developed with a computational modelling method and synthesised with cyclododecyl-2,4-dihydroxybenzoate as a "dummy" template and ethylene glycol methacrylate phosphate as a functional monomer acted as the selective recognition elements for ZON fluorescence detection. Spherical silver nanoparticles (AgNPs) were embedded in the MIP films' structure to enhance the sensor sensitivity. The imprinted films showed a high ZON recognition ability compared to non-imprinted films. Various factors that affected the measurement of the analysed sample were investigated and optimised. Embedding the AgNPs in the MIP films' structure led to an enhanced sensitivity (up to a 200-fold decrease of LOD) compared to unmodified MIP films. This fluorescent sensor system provided ZON analysis with high sensitivity, specificity, and a wider linear dynamic range of 5 ng mL-1 to 25 µg mL-1. An enhanced fluorescent sensor system based on MIP chips with embedded AgNPs could detect trace amounts of ZON in foods and feedstuffs with high sensitivity and selectivity.

Molecularly imprinted nanoparticles for pathogen visualisation.

Saccharides displayed on the cell surface of pathogens play critical roles in many activities such as adhesion, recognition and pathogenesis, as well as in prokaryotic development. In this work, we report the synthesis of molecularly imprinted nanoparticles (nanoMIPs) against pathogen surface monosaccharides using an innovative solid-phase approach. These nanoMIPs can serve as robust and selective artificial lectins specific to one particular monosaccharide. The evaluation of their binding capabilities has been implemented against bacterial cells (E. coli and S. pneumoniae) as model pathogens. The nanoMIPs were produced against two different monosaccharides: mannose (Man), which is present mainly on the surface of Gram-negative bacteria, and N-acetylglucosamine (GlcNAc) exposed on the surface of the majority of bacteria. Herein, we assessed the potential use of nanoMIPs for pathogen cell imaging and detection via flow cytometry and confocal microscopy.

Evading and overcoming AAV neutralization in gene therapy.

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.

Rapid Selective Detection and Quantification of ß-Blockers Used in Doping Based on Molecularly Imprinted Nanoparticles (NanoMIPs).

Human performance enhancing drugs (PEDs), frequently used in sport competitions, are strictly prohibited by the World Anti-Doping Agency (WADA). Biological samples collected from athletes and regular patients are continuously tested regarding the identification and/or quantification of the banned substances. Current work is focused on the application of a new analytical method, molecularly imprinted nanoparticles (nanoMIPs), to detect and determine concentrations of certain prohibited drugs, such as ß-blockers, in water and human urine samples. These medications are used in the treatment of cardiovascular conditions, negative effects of adrenaline (helping to relief stress), and hypertension (slowing down the pulse and softening the arteries). They can also significantly increase muscle relaxation and improve heart efficiency. The new method of the detection and quantification of ß-blockers is based on synthesis, characterization, and implementation of nanoMIPs (so-called plastic antibodies). It offers numerous advantages over the traditional methods, including high binding capacity, affinity, and selectivity for target molecules. Additionally, the whole process is less complicated, cheaper, and better controlled. The size and shape of the nanoMIPs is evaluated by dynamic light scattering (DLS) and transmission electron microscope (TEM). The affinity and selectivity of the nanoparticles are investigated by competitive pseudo enzyme-linked immunosorbent assay (pseudo-ELISA) similar to common immunoassays employing natural antibodies. To provide reliable results towards either doping detection or therapeutic monitoring using the minimal invasive method, the qualitative and quantitative analysis of these drugs is performed in water and human urine samples. It is demonstrated that the assay can detect ß-blockers in water within the linear range 1 nmol·L-1-1 mmol·L-1 for atenolol with the detection limit 50.6 ng mL-1, and the linear range 1 mmol·L-1-10 mmol·L-1 for labetalol with the detection limit of 90.5 ng·mL-1. In human urine samples, the linear range is recorded in the concentration range 0.1 mmol·L-1-10 nmol·L-1 for atenolol and 1 mmol·L-1-10 nmol·L-1 for labetalol with a detection limit of 61.0 ng·mL-1 for atenolol and 99.4 ng·mL-1 for labetalol.

Snapshot imprinting as a tool for surface mapping and identification of novel biomarkers of senescent cells.

Cellular senescence has proved to be a strong contributor to ageing and age-related diseases, such as cancer and atherosclerosis. Therefore, the protein content of senescent cells is highly relevant to drug discovery, diagnostics and therapeutic applications. However, current technologies for the analysis of proteins are based on a combination of separation techniques and mass spectrometry, which require handling large sample sizes and a large volume of data and are time-consuming. This limits their application in personalised medicine. An easy, quick and inexpensive procedure is needed for qualitative and quantitative analysis of proteins expressed by a cell or tissue. Here, we describe the use of the "snapshot imprinting" approach for the identification of proteins differentially expressed by senescent cells. Molecularly imprinted polymer nanoparticles (MIPs) were formed in the presence of whole cells. Following trypsinolysis, protein epitopes protected by complex with MIPs were eluted from the nanoparticles and analysed by LC-MS/MS. In this work, "snapshot imprinting" was performed parallel to a standard proteomic "shaving approach", showing similar results. The analysis by "snapshot imprinting" identified three senescent-specific proteins: cell division cycle 7-related protein kinase, partitioning defective three homolog B and putative ATP-dependent RNA helicase DHX57, the abundance of which could potentially make them specific markers of senescence. Identifying biomarkers for the future elimination of senescent cells grants the potential for developing therapeutics for age-related diseases.

Nanoparticle-induced enhancement of cholinesterase activity in the presence of malathion: A potential nerve agent therapeutic.

Organophosphate nerve agents are associated with assassination, terrorism and chemical warfare, but there has been slow progress in developing a broad-spectrum response to poisoning. For some nerve agents the oxime component of the therapy may not be effective, limiting the effectiveness of emergency treatment that is desperately needed. An alternative therapy may be possible based on accelerating enzyme (acetylcholinesterase) catalysis in unaffected adjacent enzymes. Herein we demonstrate a restoration of acetylcholinesterase activity in malathion-inhibited cell membrane preparations by the administration of functional nanoparticles. The molecularly imprinted polymer nanoparticles were designed to bind selectively to designated enzyme epitopes. Enzyme activity of membrane-bound acetylcholinesterase was measured in the presence of the organophosphate malathion and the selected nanoparticles. Enzymatic acceleration of the cholinesterase was observed at 162 ± 17 % the rate of erythrocyte ghosts without bound nanoparticles. This may restore sufficient acetylcholine hydrolysis to mitigate the effects of poisoning, offsetting the acetylcholine accumulation resulting from enzyme inhibition.

Assessing the In Vivo Biocompatibility of Molecularly Imprinted Polymer Nanoparticles.

Molecularly imprinted polymer nanoparticles (nanoMIPs) are high affinity synthetic receptors which show promise as imaging and therapeutic agents. Comprehensive analysis of the in vivo behaviour of nanoMIPs must be performed before they can be considered for clinical applications. This work reports the solid-phase synthesis of nanoMIPs and an investigation of their biodistribution, clearance and cytotoxicity in a rat model following both intravenous and oral administration. These nanoMIPs were found in each harvested tissue type, including brain tissue, implying their ability to cross the blood-brain barrier. The nanoMIPs were cleared from the body via both faeces and urine. Furthermore, we describe an immunogenicity study in mice, demonstrating that nanoMIPs specific for a cell surface protein showed moderate adjuvant properties, whilst those imprinted for a scrambled peptide showed no such behaviour. Given their ability to access all tissue types and their relatively low cytotoxicity, these results pave the way for in vivo applications of nanoMIPs.

Molecular imprinting as a tool for determining molecular markers: a lung cancer case.

Determining which cancer patients will be sensitive to a given therapy is essential for personalised medicine. Thus, it is important to develop new tools that will allow us to stratify patients according to their predicted response to treatment. The aim of work presented here was to use molecular imprinting for determining the sensitivity of lung cancer cell lines to ionising radiation based on cell surface proteomic differences. Molecularly imprinted polymer nanoparticles (nanoMIPs) were formed in the presence of whole cells. Following trypsinolysis, protein epitopes protected by complexing with MIPs were eluted from the nanoparticles and analysed by LC-MS/MS. The analysis identified two membrane proteins, neutral amino acid transporter B (0) and 4F2 cell-surface antigen heavy chain, the abundance of which in the lung cancer cells could indicate resistance of these cells to radiotherapy. This proof-of-principle experiments shows that this technology can be used in the discovery of new biomarkers and in development of novel diagnostic and therapeutic tools for a personalised medicine approach to treating cancer.

Iodo Silanes as Superior Substrates for the Solid Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles.

Current state-of-the-art techniques for the solid phase synthesis of molecularly imprinted polymer (MIP) nanoparticles typically rely on amino silanes for the immobilisation of template molecules prior to polymerisation. An investigation into commonly used amino silanes identified a number of problematic side reactions which negatively affect the purity and affinity of these polymers. Iodo silanes are presented as a superior alternative in a case study describing the synthesis of MIPs against epitopes of a common cancer biomarker, epidermal growth factor receptor (EGFR). The proposed iodo silane outperformed the amino silane by all metrics tested, showing high purity and specificity, and nanomolar affinity for the target peptide.

Nano-molecularly imprinted polymers (nanoMIPs) as a novel approach to targeted drug delivery in nanomedicine.

Molecularly imprinted polymers - MIPs - denote synthetic polymeric structures that selectively recognize the molecule of interest against which MIPs are templated. A number of works have demonstrated that MIPs can exceed the affinity and selectivity of natural antibodies, yet operating by the same principle of "lock and key". In contrast to antibodies, which have certain limitations related to the minimal size of the antigen, nanoMIPs can be fabricated against almost any target molecule irrespective of its size and low immunogenicity. Furthermore, the cost of MIP production is much lower compared to the cost of antibody production. Excitingly, MIPs can be used as nanocontainers for specific delivery of therapeutics both in vitro and in vivo. The adoption of the solid phase synthesis rendered MIPs precise reproducible characteristics and, as a consequence, improved the controlled release of therapeutic payloads. These major breakthroughs paved the way for applicability of MIPs in medicine as a novel class of therapeutics. In this review, we highlight recent advances in the fabrication of MIPs, mechanisms of controlled release from the MIPs, and their applicability in biomedical research.

Modulation of acetylcholinesterase activity using molecularly imprinted polymer nanoparticles.

Modulation of enzyme activity allows for control over many biological pathways and while strategies for the pharmaceutical design of inhibitors are well established; methods for promoting activation, that is an increase in enzymatic activity, are not. Here we demonstrate an innovative epitope mapping technique using molecular imprinting to identify four surface epitopes of acetylcholinesterase (AChE). These identified epitopes were then used as targets for the synthesis of molecularly imprinted nanoparticles (nanoMIPs). The enzymatic activity of AChE was increased upon exposure to these nanoMIPs, with one particular identified epitope nanoMIP leading to an increase in activity of 47× compared to enzyme only. The impact of nanoMIPs on the inhibited enzyme is also explored, with AChE activity recovering from 11% (following exposure to an organophosphate) to 73% (following the addition of nanoMIPs). By stabilizing the conformation of the protein rather than targeting the active site, the allosteric nature of MIP-induced reactivation suggests a new way to promote enzyme activity, even under the presence of an inhibitor. This method of enzyme activation shows promise to treat enzyme deficiency diseases or in medical emergencies where an external agent affects protein function.

Mass spectrometric detection of KRAS protein mutations using molecular imprinting.

Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.

Enhancing Antibodies' Binding Capacity through Oriented Functionalization of Plasmonic Surfaces.

Protein A has long been used in different research fields due to its ability to specifically recognize immunoglobulins (Ig). The protein derived from Staphylococcus aureus binds Ig through the Fc region of the antibody, showing its strongest binding in immunoglobulin G (IgG), making it the most used protein in its purification and detection. The research presented here integrates, for the first time, protein A to a silicon surface patterned with gold nanoparticles for the oriented binding of IgG. The signal detection is conveyed through a metal enhanced fluorescence (MEF) system. Orienting immunoglobulins allows the exposition of the fragment antigen-binding (Fab) region for the binding to its antigen, substantially increasing the binding capacity per antibody immobilized. Antibodies orientation is of crucial importance in many diagnostics devices, particularly when either component is in limited quantities.