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Diagnostic pregnancy tests are the most widely used immunoassays for home-based use. These tests employ the well-established lateral flow assay (LFA) technique, reminiscent of affinity chromatography relying on the dual action of two orthogonal anti-hCG antibodies. Immunoassays suffer from several drawbacks, including challenges in antibody manufacturing, suboptimal accuracy, and sensitivity to adverse storing conditions. Additionally, LFAs are typically designed for single use, as the LFA technique is non-reusable. An alternative to overcome these drawbacks is to leverage molecularly imprinted polymer (MIP) technology to generate polymer-based hCG-receptors and, subsequently, non-bioreceptor-based tests. Here, we report the development of MIP nanogels for hCG detection, exploiting epitopes and magnetic templates for high-yielding dispersed phase imprinting. The resulting nanogels were designed for orthogonal targeting of two immunogenic epitopes (SV and PQ) and were thoroughly characterized with respect to physical properties, binding affinity, specificity, and sensitivity. Molecular dynamics simulations indicated a pronounced conformational overlap between the templates and the epitopes in the native protein, supporting their suitability for templating cavities for hCG recognition. Quartz crystal microbalance (QCM)-based binding tests and kinetic interaction analysis by surface plasmon resonance (SPR) revealed nanomolar dissociation constants for the MIP nanogels and their corresponding template peptides and low uptake of lutenizing hormone (LH), structurally resembling to hCG. Receptor reusability was demonstrated in the multicycle SPR sensing mode using a low pH regeneration buffer. The results suggest the feasibility of using imprinted nanogels as a class of cost-effective, stable alternatives to natural antibodies for hCG detection. We foresee applications of these binders with respect to reusable pregnancy tests and other hCG-related disease diagnostics.

Stimuli‐responsive molecularly imprinted polymers (MIPs) are exciting smart materials that are gaining substantial interest within the research community due to their versatility and possible widespread applications in biosensing, biomedicine and diagnostics, as well as chromatography and separation sciences. These materials offer significant advantages as recognition materials over their biological counterparts (antibodies) because of their ease and low cost of production along with their robustness and resistance to the extremes of temperature and pH. This much needed review aims to provide an updated summary of the various stimuli‐responsive MIPs reported to date including those relying on thermo, pH, photo, biomolecule, ion, magnetic and electrical stimuli and includes their design and synthesis. The review also explores the potential applications of the stimuli‐responsive MIPs, particularly in the fields of biosensors and diagnostics, along with biological imaging, drug delivery, disease treatments and interventions and the separation of targets from complex media. The advantages and disadvantages of the current stimuli‐responsive MIPs set out in the review, allows for researchers to gather a concise understanding of these smart‐materials and should pave the way for new methods of development and real‐world applications. We believe the review is a helpful and necessary guide for the future evolution and application of stimuli‐responsive MIPs. Stimuli‐responsive molecularly imprinted polymers (MIPs) are synthetic antibodies featuring highly specific binding sites harbored in robust polymer scaffolds that are sensitive to external triggers. Recent developments in the field are here reviewed.

Efficient and site‐specific delivery of therapeutics drugs remains a critical challenge in cancer treatment. Traditional drug nanocarriers such as antibody‐drug conjugates are not generally accessible due to their high cost and can lead to serious side effects including life‐threatening allergic reactions. Here, these problems are overcome via the engineering of supramolecular agents that are manufactured with an innovative double imprinting approach. The developed molecularly imprinted nanoparticles (nanoMIPs) are targeted toward a linear epitope of estrogen receptor alfa (ERα) and loaded with the chemotherapeutic drug doxorubicin. These nanoMIPs are cost‐effective and rival the affinity of commercial antibodies for ERα. Upon specific binding of the materials to ERα, which is overexpressed in most breast cancers (BCs), nuclear drug delivery is achieved via receptor‐mediated endocytosis. Consequentially, significantly enhanced cytotoxicity is elicited in BC cell lines overexpressing ERα, paving the way for precision treatment of BC. Proof‐of‐concept for the clinical use of the nanoMIPs is provided by evaluating their drug efficacy in sophisticated three‐dimensional (3D) cancer models, which capture the complexity of the tumor microenvironment in vivo without requiring animal models. Thus, these findings highlight the potential of nanoMIPs as a promising class of novel drug compounds for use in cancer treatment. In advanced materials research, scientists aim to enhance chemotherapy, nucleic acid, and protein drug delivery using different materials, for instance, antibody‐drug conjugates, like Enhertu. These approaches can be inaccessible to many due to financial constraints or lack of expertise. This research focuses on a novel nanomaterial approach using double imprinting to improve targeted chemotherapy. These nanomaterials selectively target breast cancer cells with strong binding, low toxicity, and efficient drug release. A biomimetic scaffold‐assisted 3D model for breast cancer is developed. This cost‐effective technology can make advanced cancer treatments more accessible and affordable, potentially replacing traditional antibodies and peptides, and benefiting a wider community.

Virus recognition has been driven to the forefront of molecular recognition research due to the COVID‐19 pandemic. Development of highly sensitive recognition elements, both natural and synthetic is critical to facing such a global issue. However, as viruses mutate, it is possible for their recognition to wane through changes in the target substrate, which can lead to detection avoidance and increased false negatives. Likewise, the ability to detect specific variants is of great interest for clinical analysis of all viruses. Here, a hybrid aptamer‐molecularly imprinted polymer (aptaMIP), that maintains selective recognition for the spike protein template across various mutations, while improving performance over individual aptamer or MIP components (which themselves demonstrate excellent performance). The aptaMIP exhibits an equilibrium dissociation constant of 1.61 nM toward its template which matches or exceeds published examples of imprinting of the spike protein. The work here demonstrates that “fixing” the aptamer within a polymeric scaffold increases its capability to selectivity recognize its original target and points toward a methodology that will allow variant selective molecular recognition with exceptional affinity. A new aptamer specific for wild‐type SARS‐CoV‐2 spike protein is developed and incorporated into a molecularly‐imprinted polymer (MIP) nanoparticle. This hybrid outperforms both individual components (aptamer and MIP) with an equilibrium binding constant below that of the SARS‐CoV‐2 spike—ACE2 receptor interaction; and with clearly superior variant selectivity suggesting a new way to rapidly develop variant selective recognition nanomaterials.

Using a solid-phase molecular imprinting technique, high-affinity nanoparticles (nanoMIPs) selective for the target antibiotics, ciprofloxacin, moxifloxacin, and ofloxacin have been synthesised. These have been applied in the development of a surface plasmon resonance (SPR) sensor for the detection of the three antibiotics in both river water and milk. The particles produced demonstrated good uniformity with approximate sizes of 65.8 ± 1.8 nm, 76.3 ± 4.1 nm, and 85.7 ± 2.5 nm, and were demonstrated to have affinities of 36.2 nM, 54.7 nM, and 34.6 nM for the ciprofloxacin, moxifloxacin, and ofloxacin nanoMIPs, respectively. Cross-reactivity studies highlighted good selectivity towards the target antibiotic compared with a non-target antibiotic. Using spiked milk and river water samples, the nanoMIP-based SPR sensor offered comparable affinity with 66.8 nM, 33.4 nM, and 55.0 nM (milk) and 39.3 nM, 26.1 nM, and 42.7 nM (river water) for ciprofloxacin, moxifloxacin, and ofloxacin nanoMIPs, respectively, to that seen within a buffer standard. Estimated LODs for the three antibiotic targets in both milk and river water were low nM or below. The developed SPR sensor showed good potential for using the technology for the capture and detection of antibiotics from food and environmental samples.

Super paramagnetic iron oxide nanoparticles (SPIONs) (∼12 nm) were synthesized as the magnetic core for an imprinted polymer (MIP) shell using 4-vinylpyridine as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the cross-linker, bringing the average size up to ∼45 nm. Five targets were imprinted—the Selective Androgen Receptor Modulators (SARMs) andarine, ligandrol and RAD-140; and the steroids estradiol and gestrinone. All MMIPs produced good selectivity when loaded with a non-target molecule, with all calculated selectivity factors above the 1.2 recommended threshold and also demonstrated good affinity/capacity. The rebinding of the target molecules from a complex matrix was also explored by using spiked river water samples. The SARMs-based MMIPs were able to rebind 99.56, 87.63 and 72.78% of their target molecules (andarine, ligandrol and RAD-140, respectively), while the steroidal-based MMIPs were able to rebind 64.54 and 55.53% of their target molecules (estradiol and gestrinone, respectively) at a nominal loading of 20 ≈ μ g in 50 mg of NPs. This work highlights the potential of these bi-functional materials for trace material clean-up of complex samples and/or subsequent analysis and opens up possibilities for further simple, rapid-to-synthesise materials for targeted clean-up.

Rapid development of antibody-based therapeutics are crucial to the agenda of innovative manufacturing of macromolecular therapies to combat emergent diseases. Although highly specific, antibody therapies are costly to produce. Molecularly imprinted polymers (MIPs) constitute a rapidly-evolving class of antigen-recognition materials that act as synthetic antibodies. We report here on the virus neutralizing capacity of hydrogel-based MIPs. We produced MIPs using porcine reproductive and respiratory syndrome virus (PRRSV-1), as a model mammalian virus. Assays were performed to evaluate the specificity of virus neutralization, the effect of incubation time and MIP concentration. Polyacrylamide and N-hydroxymethylacrylamide based MIPs produced a highly significant reduction in infectious viral titer recovered after treatment, reducing it to the limit of detection of the assay. MIP specificity was tested by comparing their neutralizing effects on PRRSV-1 to the effects on the unrelated bovine viral diarrhea virus-1; no significant cross-reactivity was observed. The MIPs demonstrated effective virus neutralization in just 2.5 min and their effect was concentration dependent. These data support the further evaluation of MIPs as synthetic antibodies as a novel approach to the treatment of viral infection.

Periodontal disease affects supporting dental structures and ranks among one of the top most expensive conditions to treat in the world. Moreover, in recent years, the disease has also been linked to cardiovascular and Alzheimer’s diseases. At present, there is a serious lack of accurate diagnostic tools to identify people at severe risk of periodontal disease progression. Porphyromonas gingivalis is often considered one of the most contributing factors towards disease progression. It produces the Arg- and Lys-specific proteases Rgp and Kgp, respectively. Within this work, a short epitope sequence of these proteases is immobilised onto a magnetic nanoparticle platform. These are then used as a template to produce high-affinity, selective molecularly imprinted nanogels, using the common monomers N-tert-butylacrylamide (TBAM), N-isopropyl acrylamide (NIPAM), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). N,N-Methylene bis(acrylamide) (BIS) was used as a crosslinking monomer to form the interconnected polymeric network. The produced nanogels were immobilised onto a planar gold surface and characterised using the optical technique of surface plasmon resonance. They showed high selectivity and affinity towards their template, with affinity constants of 79.4 and 89.7 nM for the Rgp and Kgp epitope nanogels, respectively. From their calibration curves, the theoretical limit of detection was determined to be 1.27 nM for the Rgp nanogels and 2.00 nM for the Kgp nanogels. Furthermore, they also showed excellent selectivity against bacterial culture supernatants E8 (Rgp knockout), K1A (Kgp knockout), and W50-d (wild-type) strains in complex medium of brain heart infusion (BHI).

Enzymatic activity and its tight regulation are fundamental to cellular metabolism and life. While classical models of enzyme kinetics explain the behavior of enzymes in dilute buffer solutions, there are elusive properties that emerge from enzymes in their native, crowded environments. In this study, we harness liquid-liquid phase separation (LLPS) to create controlled droplets that mimic cytosolic protein crowding, offering a unique system to understand enzyme kinetics in complex microenvironments. We uncover a mechanism in which enzyme-induced changes in shear viscosity arise from dynamic interactions among the substrate, product, and the protein crowder. Using fluorescence microscopy, bulk shear rheometry and microrheology, we show that enzymatic activity modifies the apparent viscosity of both protein-rich droplets and the surrounding PEG-rich phase, enhancing substrate mobility and improving substrate access to catalytic sites. Our findings suggest that this enzymatic-viscosity coupling affects substrate availability and influences the organization and dynamics of macromolecular crowding within droplets. These results provide new insights into how enzymes impact both their physical environment and metabolic processes in the cell.

Peptides are important biomarkers for a range of diseases, however distinguishing different amino-acid sequences using artificial receptors remains a major challenge in biomedical sensing. Here we present a new approach to creating highly selective recognition surfaces using phage display biopanning against metal-organic nanosheets (MONs) and demonstrate their use as the next-generation of biomolecular recognition surfaces. Three MONs (ZIF-7, ZIF-7-NH2 and Hf-BTB-NH2) were chosen as initial targets to demonstrate how simple synthetic modifications can enhance selectivity towards specific amino acid sequences. Each MON system was added to a solution containing every possible combination of 7-residue peptides attached to bacteriophage hosts and the highest affinity binding peptides for each system was identified via successive biopanning rounds. In each case only a single peptide sequence was isolated (YNYRNLL – ZIF-7, NNWWAPA – ZIF-7-NH2 and FTVRDLS – Hf-BTB-NH2). This indicates that these MONs are highly selective, which is attributed to their 2D nanosheet structure. Zeta potential and contact angle measurements were conducted on each MON and combined with calculated properties for the peptide sequences and binding studies to provide insights into the relative importance of electrostatic, hydrophobic and co-ordination bonding interactions. A quartz crystal microbalance (QCM) was used to model phage binding and the Hf-BTB-NH2 MON coated QCM produced a 5-fold higher signal for FTVRDLS functionalised phage compared to phage with generic peptide sequences. Further studies focusing on Hf-BTB-NH2 confirmed that the VRDL sequence was highly conserved, and on-target binding exhibited equilibrium dissociation constants that are comparable to natural recognition materials. Surface plasmon resonance (SPR) studies indicated a 4600-fold higher equilibrium dissociation constant (KD) for FTVRDLS compared to those obtained for off-target sequences, comparable to those of antibodies (KD = 4 x10-10). We anticipate that the highly tunable nature of MONs will enhance our understanding of binding interactions and enable molecular recognition of biomedically important peptides.