Molecularly imprinted nanogels as synthetic recognition materials for the ultrasensitive detection of periodontal disease biomarkers.

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).

A molecularly imprinted polymer nanoparticle-based surface plasmon resonance sensor platform for antibiotic detection in river water and milk.

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.

Green synthesis as a simple and rapid route to protein modified magnetic nanoparticles for use in the development of a fluorometric molecularly imprinted polymer-based assay for detection of myoglobin.

We have developed a low-cost molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify myoglobin in a biological sample. The assay uses a previously unreported method for the development of microwave-assisted rapid synthesis of aldehyde functionalized magnetic nanoparticles, in just 20 min. The aldehyde functionalized nanoparticles have an average size of 7.5 nm ± 1.8 and saturation magnetizations of 31.8 emu g-1 with near-closed magnetization loops, confirming their superparamagnetic properties. We have subsequently shown that protein tethering was possible to the aldehyde particles, with 0.25 ± 0.013 mg of myoglobin adsorbed to 20 mg of the nanomaterial. Myoglobin-specific fluorescently tagged MIP (F-MIP) particles were synthesized and used within the assay to capture myoglobin from a test sample. Excess F-MIP was removed from the sample using protein functionalized magnetic nanoparticles (Mb-SPION), with the remaining sample analyzed using fluorescence spectroscopy. The obtained calibration plot of myoglobin showed a linear correlation ranging from 60 pg ml-1 to 6 mg ml-1 with the limit of detection of 60 pg ml-1. This method was successfully used to detect myoglobin in spiked fetal calf serum, with a recovery rate of more than 93%.

Utilisation of molecularly imprinting technology for the detection of glucocorticoids for a point of care surface plasmon resonance (SPR) device.

Herein, we describe the synthesis and characterisation of four synthetic recognition materials (nanoMIPs) selective for the glucocorticoid steroids - prednisolone, prednisone, dexamethasone, and cortisone. Using a solid-phase synthesis approach, these materials were then applied in the development of a surface plasmon resonance (SPR) sensor for the detection of these four targets in doped urine, to mimic the routine testing of agricultural waste for possible environmental exposure. The synthesised particles displayed a range of sizes between 104 and 160 nm. Affinity studies were performed, and these synthetic materials were shown to display nanomolar affinities (15.9-62.8 nM) towards their desired targets. Furthermore, we conducted cross-reactivity studies to assess the materials selectivity towards their desired target and the materials showed excellent selectivity when compared to the non-desired target, with selectivity factors calculated. Furthermore, through the use of 3D visualisation it can be seen that small changes between structures (such as a hydroxyl to ketone transformation) there is excellent selectivity between the compounds in the ranges of 100 fold plus. Using Surine™ doped samples the materials offered comparable nanomolar affinities (10.7-75.7 nM) towards their targets when compared to the standardised buffer preparation. Detection levels in urine for all compounds was in the nanomolar range. The developed sensor offers potential for these devices to be used in the prevention of these pharmaceutical compounds to enter the surrounding environment through agricultural waste through monitoring at source. Likewise, they can be used to monitor use in clinical samples.

A rapid synthesis of molecularly imprinted polymer nanoparticles for the extraction of performance enhancing drugs (PIEDs).

It is becoming increasingly more significant to detect and separate hormones from water sources, with the development of synthetic recognition materials becoming an emerging field. The delicate nature of biological recognition materials such as the antibodies means the generation of robust viable synthetic alternatives has become a necessity. Molecularly imprinted nanoparticles (NanoMIPs) are an exciting class that has shown promise due the generation of high-affinity and specific materials. While nanoMIPs offer high affinity, robustness and reusability, their production can be tricky and laborious. Here we have developed a simple and rapid microwaveable suspension polymerisation technique to produce nanoMIPs for two related classes of drug targets, Selective Androgen Receptor Modulators (SARMs) and steroids. These nanoMIPs were produced using one-pot microwave synthesis with methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as a suitable cross-linker, producing particles of an approximate range of 120-140 nm. With the SARMs-based nanoMIPs being able to rebind 94.08 and 94.46% of their target molecules (andarine, and RAD-140, respectively), while the steroidal-based nanoMIPs were able to rebind 96.62 and 96.80% of their target molecules (estradiol and testosterone, respectively). The affinity of nanoMIPs were investigated using Scatchard analysis, with Ka values of 6.60 × 106, 1.51 × 107, 1.04 × 107 and 1.51 × 107 M-1, for the binding of andarine, RAD-140, estradiol and testosterone, respectively. While the non-imprinted control polymer (NIP) shows a decrease in affinity with Ka values of 3.40 × 104, 1.01 × 104, 1.83 × 104, and 4.00 × 104 M-1, respectively. The nanoMIPs also demonstrated good selectivity and specificity of binding the targets from a complex matrix of river water, showing these functional materials offer multiple uses for trace compound analysis and/or sample clean-up.

Highly Selective Aptamer-Molecularly Imprinted Polymer Hybrids for Recognition of SARS-CoV-2 Spike Protein Variants.

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.

Detection of selective androgen receptor modulators (SARMs) in serum using a molecularly imprinted nanoparticle surface plasmon resonance sensor.

Selective Androgen Receptor Modulators (SARMs) are a fairly new class of therapeutic compounds that act upon the androgen receptor. They proffer similar anabolic properties to steroids, but with a much-reduced androgenic profile. They have become a popular substance of abuse in competitive sport. Being relatively new, detection systems are limited to chromatographic methods. Here we present a surface plasmon resonance sensor for three commonly-used SARMS, Andarine, Ligandrol and RAD-140, using high-affinity molecularly imprinted nanoparticles (nanoMIPs) as the recognition element. Synthesised nanoMIPS exhibited dissociation constant (KD) values of 29.3 nM, 52.5 nM and 75.1 nM for Andarine, Ligandrol and RAD-140 nanoMIPs, respectively. Cross-reactivity of the particles was explored using the alternative SARMs, with the nanoMIPs demonstrating good specificity. Fetal Bovine Serum (FBS) was used to assess the ability of the SPR-based nanoMIP sensor to detect the target compounds in a comparable biological matrix, with observed KD values of 12.3 nM, 31.9 nM and 28.1 nM for Andarine, Ligandrol and RAD-140 nanoMIPs, respectively. Theoretical limits of detection (LoD) were estimated from a calibration plot in FBS and show that the nanoMIP-based sensors have the potential to theoretically measure these SARMs in the low to sub nM range. Crucially these levels are below the minimum required performance limit (MRPL) set for these compounds by WADA. This study highlights the power of modern molecular imprinting to rapidly address required molecular recognition for new compounds of interest.

Application of thymine-based nucleobase-modified acrylamide as a functional co-monomer in electropolymerised thin-film molecularly imprinted polymer (MIP) for selective protein (haemoglobin) binding.

Molecularly imprinted polymers (MIPs) are fast becoming alternatives to biological recognition materials, offering robustness and the ability to work in extreme environments. Here, a modified thymine-based nucleobase, with acrylamide at the 5-postion (AA-dT) was used as a co-monomer in the synthesis of a thin-film electropolymerised MIP system for the molecular recognition of the protein haemoglobin. The AA-dT co-monomer incorporated into a N-hydroxymethylacrylamide (NHMAm) MIP offered a two-fold superior binding affinity of the NHMAm only MIP, with KD values of 0.72 µM and 1.67 µM, respectively. A unique AA-dT:NHMAm MIP bilayer was created in an attempt to increase the amount AA-dT incorporated into the film, and this obtained a respectable KD value of 7.03 µM. All MIPs produced excellent selectivity for the target protein and when applied to a sensor platform (Surface Plasma Resonance), the limit of detection for the MIPs is in the nM range (3.87, 3.47, and 3.87 nM, for the NHMAm MIP, AA-dT:NHMAm MIP, and AA-dT:NHMAm MIP bilayer, respectively). The introduction of the modified thymine-based nucleobase offers a promising strategy for improving the properties of a MIP, allowing these MIPs to potentially be a highly robust and selective material for molecular recognition.

Evaluation of acrylamide-based molecularly imprinted polymer thin-sheets for specific protein capture-a myoglobin model.

We evaluate a series of thin-sheet hydrogel molecularly imprinted polymers (MIPs), using a family of acrylamide-based monomers, selective for the target protein myoglobin (Mb). The simple production of the thin-sheet MIP offers an alternative biorecognition surface that is robust, stable and uniform, and has the potential to be adapted for biosensor applications. The MIP containing the functional monomerN-hydroxymethylacrylamide (NHMAm), produced optimal specific rebinding of the target protein (Mb) with 84.9% (± 0.7) rebinding and imprinting and selectivity factors of 1.41 and 1.55, respectively. The least optimal performing MIP contained the functional monomerN,N-dimethylacrylamide (DMAm) with 67.5% (± 0.7) rebinding and imprinting and selectivity factors of 1.11 and 1.32, respectively. Hydrogen bonding effects, within a protein-MIP complex, were investigated using computational methods and Fourier transform infrared (FTIR) spectroscopy. The quantum mechanical calculations predictions of a red shift of the monomer carbonyl peak is borne-out within FTIR spectra, with three of the MIPs, acrylamide, N-(hydroxymethyl) acrylamide, andN-(hydroxyethyl) acrylamide, showing peak downshifts of 4, 11, and 8 cm-1, respectively.

Hybrid Aptamer-Molecularly Imprinted Polymer (aptaMIP) Nanoparticles from Protein Recognition-A Trypsin Model.

Aptamers offer excellent potential for replacing antibodies for molecular recognition purposes however their performance can compromise with biological/environmental degradation being a particular problem. Molecularly imprinted Polymers (MIPs) offer an alternative to biological materials and while these offer the robustness and ability to work in extreme environmental conditions, they often lack the same recognition performance. By slightly adapting the chemical structure of a DNA aptamer it is incorporated for use as the recognition part of a MIP, thus creating an aptamer-MIP hybrid or aptaMIP. Here these are developed for the detection of the target protein trypsin. The aptaMIP nanoparticles offer superior binding affinity over conventional MIP nanoparticles (nanoMIPs), with KD values of 6.8 × 10-9 (±0.2 × 10-9 ) m and 12.3 × 10-9 (±0.4 × 10-9 ) m for the aptaMIP and nanoMIP, respectively. The aptaMIP also outperforms the aptamer only (10.3 × 10-9 m). Good selectivity against other protein targets is observed. Using surface plasmon resonance, the limit of detection for aptaMIP nanoparticles is twofold lower (2 nm) compared to the nanoMIP (4 nm). Introduction of the aptamer as a "macro-monomer" into the MIP scaffold has beneficial effects and offers potential to improve this class of polymers significantly.

Toward Rational Design of Selective Molecularly Imprinted Polymers (MIPs) for Proteins: Computational and Experimental Studies of Acrylamide Based Polymers for Myoglobin.

Molecularly imprinted polymers (MIPs) have potential as alternatives to antibodies in the diagnosis and treatment of disease. However, atomistic level knowledge of the prepolymerization process is limited that would facilitate rational design of more efficient MIPs. Accordingly, we have investigated using computation and experiment the protein-monomer binding interactions that may influence the desired specificity. Myoglobin was used as the target protein and five different acrylamide-based monomers were considered. Protein binding sites were predicted using SiteMap and binding free energies of monomers at each site were calculated using MM-GBSA. Statistical thermodynamic analysis and study of atomistic interactions facilitated rationalization of monomer performance in MIP rebinding studies (% rebind; imprinting factors). CD spectroscopy was used to determine monomer effects on myoglobin secondary structure, with all monomers except the smallest monomer (acrylamide) causing significant changes. A complex interplay between different protein-monomer binding effects and MIP efficacy was observed. Validation of hypotheses for key binding features was achieved by rational selection of two different comonomer MIP combinations that produced experimental results in agreement with predictions. The comonomer studies revealed that uniform, noncompetitive binding of monomers around a target protein is favorable. This study represents a step toward future rational in silico design of MIPs for proteins.

Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody Mimics.

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.