Nanoplasmonic biosensor for rapid detection of multiple viral variants in human serum.

As viruses constantly change due to mutation, variants are expected to emerge demanding development of sensors capable of detecting multiple variants using one single sensor platform. Herein, we report the integration of a synthetic binder against SARS-CoV-2 with a nanoplasmonic-based sensing technology, which enables the successful detection of spike proteins of Alpha, Beta and Gamma variants of SARS CoV-2. The recognition event is achieved by specific nanostructured molecularly imprinted polymers (nanoMIPs), developed against a region of the receptor binding domain (RBD) of the SARS CoV-2 spike protein. The transduction is based on the principle of localized surface plasmon resonance (LSPR) associated with silver nanostructures. The nanoMIPs-functionalised LSPR sensor allows for the detection of all 3 protein variants with a limit of detection of 9.71 fM, 7.32 fM and 8.81 pM using wavelength shifts respectively for Alpha, Beta and Gamma spike protein variants. This can be achieved within 30 min from the sample collection, both from blood and using nasal swab, thus making this sensor suitable for rapid detection of COVID-19. Additionally, the turnaround time for sensor development and validation can be completed in less than 8 weeks, making it suitable for addressing future pandemic needs without the requirement for biological binding agents, which is one of the bottlenecks to the supply chain in diagnostic devices.

Probing Peptide Sequences on Their Ability to Generate Affinity Sites in Molecularly Imprinted Polymers.

An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

NanoMIP-based approach for the suppression of interference signals in electrochemical sensors.

Herein, we describe the use of molecularly imprinted nanoparticles (nanoMIPs) as sequestering (masking) agents, to suppress the signal coming from interfering molecules and facilitate the detection of the target analyte. In this work, ascorbic acid was used as a model interfering molecule in dopamine electrochemical detection. NanoMIPs selective for ascorbic acid demonstrated to be capable of binding and suppressing electrochemical signal from ascorbic acid, enabling the detection of dopamine in the range 100-500 nM, without any need for sample pre-treatment. Tests in real samples (spiked human serum) were also carried out successfully. Due to the generic nature of the imprinting process, the proposed approach can be tailored to suppress potentially any interfering species, by simply varying the type of nanoMIPs used.

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.

Molecularly imprinted polymer nanoparticle-based assay (MINA): application for fumonisin B1 determination.

The enzyme-linked immunosorbent assay (ELISA) has been used as a standard tool for monitoring food and animal feed contamination from the carcinogenic fumonisin B1 (FB1). Unfortunately, ELISA is not always efficient due to the instability of the antibody and enzyme components in the immunoassay, the presence of natural enzyme inhibitors in the samples and the high levels of non-specific protein binding. Additionally, the production of antibodies for ELISA can be time-consuming and costly, due to the involvement of animals in the manufacturing process. To overcome these limiting factors, a molecularly imprinted nanoparticle based assay (MINA) has been developed, where the molecularly imprinted nanoparticles (nanoMIPs) replace the primary antibody used in a competitive ELISA. Herein, computational modelling was used to design the nanoMIPs by selecting monomers that specifically interact with FB1. The affinity of the monomers to FB1 was verified by measuring their binding in affinity chromatography experiments. The nanoMIPs were produced by solid phase synthesis and the results showed that nanoMIPs had a hydrodynamic diameter of around 249 ± 29 nm. The assay tested in model samples is highly selective and does not show cross-reactivity with other mycotoxins such as fumonisin B2 (FB2), aflatoxin B1 (AFB1), citrinin (CTT), zearalenone (ZEA), and deoxynivalenol (DON). The MINA allows the detection of FB1 in the concentration range of 10 pM-10 nM with a detection limit of 1.9 pM and a recovery of 108.13-113.76%.

In Vivo Recognition of Human Vascular Endothelial Growth Factor by Molecularly Imprinted Polymers.

One of the mechanisms responsible for cancer-induced increased blood supply in malignant neoplasms is the overexpression of vascular endothelial growth factor (VEGF). Several antibodies for VEGF targeting have been produced for both imaging and therapy. Molecularly imprinted polymer nanoparticles, nanoMIPs, however, offer significant advantages over antibodies, in particular in relation to improved stability, speed of design, cost and control over functionalization. In the present study, the successful production of nanoMIPs against human VEGF is reported for the first time. NanoMIPs were coupled with quantum dots (QDs) for cancer imaging. The composite nanoparticles exhibited specific homing toward human melanoma cell xenografts, overexpressing hVEGF, in zebrafish embryos. No evidence of this accumulation was observed in control organisms. These results indicate that nanoMIPs are promising materials which can be considered for advancing molecular oncological research, in particular when antibodies are less desirable due to their immunogenicity or long production time.

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%.

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.

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.

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.

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.

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.

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.

Molecularly Imprinted Polymer Nanoparticles Enable Rapid, Reliable, and Robust Point-of-Care Thermal Detection of SARS-CoV-2.

Rapid antigen tests are currently used for population screening of COVID-19. However, they lack sensitivity and utilize antibodies as receptors, which can only function in narrow temperature and pH ranges. Consequently, molecularly imprinted polymer nanoparticles (nanoMIPs) are synthetized with a fast (2 h) and scalable process using merely a tiny SARS-CoV-2 fragment (∼10 amino acids). The nanoMIPs rival the affinity of SARS-CoV-2 antibodies under standard testing conditions and surpass them at elevated temperatures or in acidic media. Therefore, nanoMIP sensors possess clear advantages over antibody-based assays as they can function in various challenging media. A thermal assay is developed with nanoMIPs electrografted onto screen-printed electrodes to accurately quantify SARS-CoV-2 antigens. Heat transfer-based measurements demonstrate superior detection limits compared to commercial rapid antigen tests and most antigen tests from the literature for both the alpha (∼9.9 fg mL-1) and delta (∼6.1 fg mL-1) variants of the spike protein. A prototype assay is developed, which can rapidly (∼15 min) validate clinical patient samples with excellent sensitivity and specificity. The straightforward epitope imprinting method and high robustness of nanoMIPs produce a SARS-CoV-2 sensor with significant commercial potential for population screening, in addition to the possibility of measurements in diagnostically challenging environments.

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.

Carboxyl-fentanyl detection using optical fibre grating-based sensors functionalised with molecularly imprinted nanoparticles.

Butyrylfentanyl is a new designer drug reported with growing use and related deaths. Routine toxicological analyses of this novel synthetic opioid drug have not been established yet. This work reports a fibre optic sensor that measures carboxyl-fentanyl which is the major metabolite of butyrylfentanyl presented in blood, providing a promising tool for detecting butyrylfentanyl intoxication. A long period fibre grating (LPG) sensor array operating at phase-matching condition is deployed in combination with a state-of-the-art molecular imprinting technique. Nano-sized molecularly imprinted polymers (nanoMIPs) are synthesised via a solid-phase approach and coated on the surface of an LPG array. An LPG array consists of two parts: a detection and a reference LPG. The former is functionalised with nanoMIPs prior to the measurements, whilst the latter is used to take into account the temperature response of the detection LPG. The developed sensor exhibits a gradual response over increasing concentrations of carboxyl-fentanyl from 0 to 1000 ng/mL with a minimal detected concentration of 50 ng/mL, that corresponds to a wavelength shift of 1.20 ± 0.2 nm. The Langmuir adsorption isotherm is applied to fit the analytical data which reveal a binding constant of 2.03 µM-1. The developed sensor shows high selectivity in detecting carboxyl-fentanyl among other drugs and potential interferents including morphine, cocaine, glucose and albumin. It shows a certain degree of cross-response to fentanyl which shares the same binding sites as carboxyl-fentanyl and therefore can be potentially used to detect fentanyl.

Immobilization of Molecularly Imprinted Polymer Nanoparticles onto Surfaces Using Different Strategies: Evaluating the Influence of the Functionalized Interface on the Performance of a Thermal Assay for the Detection of the Cardiac Biomarker Troponin I.

We demonstrate that a novel functionalized interface, where molecularly imprinted polymer nanoparticles (nanoMIPs) are attached to screen-printed graphite electrodes (SPEs), can be utilized for the thermal detection of the cardiac biomarker troponin I (cTnI). The ultrasensitive detection of the unique protein cTnI can be utilized for the early diagnosis of myocardial infraction (i.e., heart attacks), resulting in considerably lower patient mortality and morbidity. Our developed platform presents an innovative route to develop accurate, low-cost, and disposable sensors for the diagnosis of cardiovascular diseases, specifically myocardial infraction. A reproducible and advantageous solid-phase approach was utilized to synthesize high-affinity nanoMIPs (average size = 71 nm) for cTnI, which served as synthetic receptors in a thermal sensing platform. To assess the performance and commercial potential of the sensor platform, various approaches were used to immobilize nanoMIPs onto thermocouples or SPEs: dip coating, drop casting, and a covalent approach relying on electrografting with an organic coupling reaction. Characterization of the nanoMIP-functionalized surfaces was performed with electrochemical impedance spectroscopy, atomic force microscopy, and scanning electron microscopy. Measurements from an in-house designed thermal setup revealed that covalent functionalization of nanoMIPs onto SPEs led to the most reproducible sensing capabilities. The proof of application was provided by measuring buffered solutions spiked with cTnI, which demonstrated that through monitoring changes in heat transfer at the solid-liquid interface, we can measure concentrations as low as 10 pg L-1, resulting in the most sensitive test of this type. Furthermore, preliminary data are presented for a prototype platform, which can detect cTnI with shorter measurement times and smaller sample volumes. The excellent sensor performance, versatility of the nanoMIPs, and reproducible and low-cost nature of the SPEs demonstrate that this sensor platform technology has a clear commercial route with high potential to contribute to sustainable healthcare.

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.

Generation of High-Affinity Molecularly Imprinted Nanoparticles for Protein Recognition via a Solid-Phase Synthesis Protocol.

Molecularly imprinted polymers are leading technology in the development of protein biomimetics. This chapter describes the protocol for the synthesis of protein imprinted nanoparticles. These materials exhibit exceptional affinity (into the nM/pM range) and selectivity for their target template. The nanoparticles can be developed for a wide range of targets, while exhibiting excellent robustness, solubility, and flexibility in use. They are finding use in the creation of drug delivery vectors and sensing and recognition assays.

Functionalized Core-Shell Yttrium Oxide Nanoparticles as Antioxidants Agents in Heat Stressed Rats.

Herein, we assess the antioxidant potential of core-shell yttrium oxide nanoparticles functionalized with ethylene glycol methacrylate phosphate (EGMP). The antioxidant properties of these nanoparticles were investigated in heat stressed models of 21 rats (heat stressed group, group B). Different samples of blood, serum, and tissue homogenate were collected at different time intervals in order to measure oxidative biomarkers such as enzymatic antioxidants (SODs, GPX, GST, GR, and TAC) and oxidative byproducts (MDA, PC, and 8-OHdG). Liver specimens of prophylactic group and heat stressed ones were also histopathologically examined 2 h post NPs injection. The measurements of oxidative biomarkers were complementary with histopathological findings and confirmed the antioxidant properties of poly EGMP yttrium oxide NPs.