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.

Detection of multiple steroidal compounds in synthetic urine using comprehensive gas chromatography-mass spectrometry (GC×GC-MS) combined with a molecularly imprinted polymer clean-up protocol.

A method capable of screening for multiple steroids in urine has been developed, using a series of twelve structurally similar, and commercially relevant compounds as target analytes. A molecularly imprinted solid phase extraction clean-up step was used to make the sample suitable for injection onto a GC×GC-MS setup. Significant improvements compared to a commercially available C-18 material were observed. Each individual steroid was able to be separated and identified, using both the retention profile and diagnostic fragmentation ion monitoring abilities of the comprehensive chromatographic-mass spectrometry method. Effective LODs of between 11.7 and 27.0 pg were calculated for individual steroids, effectively equivalent to concentration levels of between 0.234 and 0.540 ng mL(-1) in urine, while the application of multiple screen was demonstrated using a 10 ng mL(-1) mixed sample. The nature of this study also removes the need for sample derivitisation which speeds up the screening process.

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.

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.

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.

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.

Formation of protein molecular imprints within Langmuir monolayers: a quartz crystal microbalance study.

Protein imprinting leading to enhanced rebinding of ferritin to ternary lipid monolayers is demonstrated using a quartz crystal microbalance. Monolayers consisting of cationic dioctadecyldimethylammonium bromide, non-ionic methyl stearate, and poly(ethylene glycol) bearing phospholipids were imprinted with ferritin at the air/water interface of a Langmuir-Blodgett trough and transferred hydrated to hydrophobic substrates for study. This immobilization was shown by fluorescence correlation spectroscopy to significantly hinder any further diffusion of lipids, while rebinding studies demonstrated up to a six-fold increase in ferritin adsorption to imprinted versus control monolayers. A diminished rebinding of ferritin to its imprint was observed through pH reduction to below the protein isoelectric point, demonstrating the electrostatic nature of the interaction. Rebinding to films where imprint pockets remained occupied by the template protein was also minimal. Studies with a smaller acidic protein revealed the importance of the steric influence of poly(ethylene glycol) in forming the protein binding pockets, as albumin-imprinted monolayers showed low binding of ferritin, while ferritin-imprinted monolayers readily accommodated albumin. The controllable structure-function relationship and limitations of this system are discussed with respect to the application of protein imprinting in sensor development as well as fundamental studies of proteins at dynamic interfaces.

From 3D to 2D: a review of the molecular imprinting of proteins.

Molecular imprinting is a generic technology that allows for the introduction of sites of specific molecular affinity into otherwise homogeneous polymeric matrices. Commonly this technique has been shown to be effective when targeting small molecules of molecular weight <1500, while extending the technique to larger molecules such as proteins has proven difficult. A number of key inherent problems in protein imprinting have been identified, including permanent entrapment, poor mass transfer, denaturation, and heterogeneity in binding pocket affinity, which have been addressed using a variety of approaches. This review focuses on protein imprinting in its various forms, ranging from conventional bulk techniques to novel thin film and monolayer surface imprinting approaches.

Molecularly imprinted polymers in clinical diagnostics--future potential and existing problems.

The last five years have witnessed a fast progress in the area of molecularly imprinted polymers (MIPs). These have included the development of rational protocols for polymer design (combinatorial and computational), the development of MIPs compatible for use in aqueous environment and the development of various procedures for the integration of MIPs with sensors. The substantial improvements in the performance of imprinted polymers have also been accompanied by a growing number of MIP publications related to solving practical problems associated with their use, e.g. in environmental and clinical analysis. This paper furnishes a detailed analysis of recent achievements in MIPs design and applications related to healthcare, made by our group as well as others worldwide.

Polythiophene nanofilms for sensitive fluorescence detection of viruses in drinking water.

Molecular imprints of the tobacco necrosis virus (TNV) have been formed within polythiophene nanofilms with an approximate thickness of 200nm. These films have been electrochemically deposited onto conducting Au surfaces. Upon rebinding, the TNV-polythiophene complex changes the fluorescence intensity of the nanofilm. The fluorescence intensity at 410nm was observed to be proportional to the concentration of viruses in the range of 0.1-10ngL(-1) (0.15-15pg) with the lower calculated detection limit of 2.29ngL(-1) (3.4pg). The intensity of the fluorescence emission is not affected by the thickness of the polythiophene film and the nature of TNV specific binding sites. Kinetic data analyses showed that the nanofilm responds to TNV within 2min; and cross-selectivity studies with tobacco mosaic virus (TMV) showed an excellent specificity for the targeted TNV. These binding experiments demonstrate the potential of fluorescence emission for the specific, label free and rapid detection of viruses using nanofilm sensors. Taking into account the lower limit of detection, the fluorescence sensing reported here is reliable, simple to perform, rapid, cost-effective and offers a sensitive analytical method for virus detection in water resources.

Controlled release of the herbicide simazine from computationally designed molecularly imprinted polymers.

The present study describes the development of materials suitable for environmental control of algae. Molecularly imprinted polymers (MIPs) were used as simazine carriers able to provide the controlled release of simazine into water. Three polymers were designed using computational modelling. The selection of methacrylic acid (MA) and hydroxyethyl methacrylate (HEM) as functional monomers was based on results obtained using the Leapfrog algorithm. A cross-linked polymer made without functional monomers was also prepared and tested as a control. The release of simazine from all three polymers was studied. It was shown that the presence of functional monomers is important for polymer affinity and for controlled release of herbicide. The speed of release of herbicide correlated with the calculated binding characteristics. The high-affinity MA-based polymer released approximately 2% and the low-affinity HEM-based polymer released approximately 27% of the template over 25 days. The kinetics of simazine release from HEM-based polymer show that total saturation of an aqueous environment could be achieved over a period of 3 weeks and this corresponds to the maximal simazine solubility in water. The possible use of these types of polymers in the field of controlled release is discussed.

Analytical methods for determination of mycotoxins: An update (2009-2014).

Mycotoxins are a problematic and toxic group of small organic molecules that are produced as secondary metabolites by several fungal species that colonise crops. They lead to contamination at both the field and postharvest stages of food production with a considerable range of foodstuffs affected, from coffee and cereals, to dried fruit and spices. With wide ranging structural diversity of mycotoxins, severe toxic effects caused by these molecules and their high chemical stability the requirement for robust and effective detection methods is clear. This paper builds on our previous review and summarises the most recent advances in this field, in the years 2009-2014 inclusive. This review summarises traditional methods such as chromatographic and immunochemical techniques, as well as newer approaches such as biosensors, and optical techniques which are becoming more prevalent. A section on sampling and sample treatment has been prepared to highlight the importance of this step in the analytical methods. We close with a look at emerging technologies that will bring effective and rapid analysis out of the laboratory and into the field.

Generation of novel hybrid aptamer-molecularly imprinted polymeric nanoparticles.

A strategy to exploit aptamers as recognition elements of molecularly imprinted polymeric nanoparticles (AptaMIP NPs) is presented, via modification of the chemical structure of the DNA. It is demonstrated that the introduction of this modified "aptamer monomer" results in an increase of the affinity of the produced MIP NPs, without altering their physical properties such as size, shape, or dispersibility.

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography-mass spectrometry.

A proof of principle gas chromatography-mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36-68% for PDIMs, allowing some superior detection of the target analytes.

Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A.

A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.