Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)

Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 mM, 44 ± 3 mM, 17 ± 2 mM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface

Selective extraction of proteins and other macromolecules from biological samples using molecular imprinted polymers

The accurate determination of intact macromolecules in biological samples, such as blood, plasma, serum, urine, tissue and feces is a challenging problem. The increased interest in macromolecules both as candidate drugs and as biomarkers for diagnostic purposes means that new method development approaches are needed. This review charts developments in the use of molecularly imprinted polymers first for small-molecular-mass compounds then for proteins and other macromolecules. Examples of the development of molecularly imprinted polymers for macromolecules are highlighted. The two main application areas to date are sensors and separation science, particularly SPE. Examples include peptides and polypeptides, lysozyme, hemoglobin, ovalbumin, bovine serum albumin and viruses

Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620–800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork–venison, pork–shad and venison–shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml − 1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species

Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs

We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-hydroxymethylacrylamide, NHMA; N-isopropylacrylamide, NiPAm) for their capability of imprinting protein. Rebinding capacities (Q) from spectroscopic studies were highest for bovine haemoglobin (BHb) MIPs based on AA, Q = 4.8 ± 0.21 76 ± 0.5%). When applied to the QCM sensor as thin-film MIPs, NHMA MIPs were found to exhibit best discrimination between MIP and non-imprinted control polymer (NIP) in the order of NiPAm < AA < NHMA. The extent of template removal and rebinding, using both crystal impedance and frequency measurements, demonstrated that 10% (w/v):10% (v/v) sodium dodecyl sulphate:acetic acid (pH 2.8) was efficient at eluting template BHb (with 80 ± 10% removal). Selectivity studies of NHMA BHb-MIPs revealed higher adsorption and selective recognition properties to BHb (64.5 kDa) when compared to non-cognate BSA (66 kDa), myoglobin (Mb, 17.5 kDa), lysozyme (Lyz, 14.7 kDa) thaumatin (Thau, 22 kDa) and trypsin (Tryp, 22.3 kDa). The QCM gave frequency shifts of ∼1500 ± 50 Hz for template BHb rebinding in both AA and NHMA MIPs, whereas AA-based MIPs exhibited an interference signal of ∼2200 ± 50 Hz for non-cognate BSA in comparison to a ∼500 ± 50 Hz shift with NHMA MIPs. Our results show that NHMA-based hydrogel MIP are superior to AA and NIPAM

MIP-based electrochemical protein profiling

We present the development of an electrochemical biosensor based on modified glassy carbon (GC) electrodes using hydrogel-based molecularly imprinted polymers (MIPs) has been fabricated for protein detection. The coupling of pattern recognition techniques via principal component analysis (PCA) has resulted in unique protein fingerprints for corresponding protein templates, allowing for MIP-based protein profiling. Polyacrylamide MIPs for memory imprinting of bovine haemoglobin (BHb), equine myoglobin (EMb), cytochrome C (Cyt C), and bovine serum albumin (BSA), alongside a non-imprinted polymer (NIP) control, were spectrophotometrically, and electrochemically characterised using modified GC electrodes. Rebinding capacities (Q) were revealed to be higher for larger proteins (BHb and BSA, Q ≈ 4.5) while (EMb and Cyt C, Q ≈ 2.5). Electrochemical results show that due to the selective nature of MIPs, protein arrival at the electrode via diffusion is delayed, in comparison to a NIP, by attractive selective interactions with exposed MIP cavities. However, at lower concentrations such discriminations are difficult due to low levels of MIP rebinding. PCA loading plots revealed 5 variables responsible for the separation of the proteins; Ep, Ip, E1/2 , Iat −0.8 V, �Idecay peak current to −0.8 V. Statistical symmetric measures of agreement using Cohen’s kappa coefficient (K) were revealed to be 63% for bare GC, 96% for NIP and 100% for MIP. Therefore, our results show that with the use of PCA such discriminations are achievable, also with the advantage of faster detection rates. The possibilities for this MIP technology once fully developed are vast, including uses in bio-sample clean-up or selective extraction, replacement of biological antibodies in immunoassays, as well as biosensors for medicine, food and the environment

Automating the application of smart materials for protein crystallization

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as 'smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials. © 2015, IUCR. All rights reserved

Effectiveness of and public perception about the installation of transverse rumble strips with road studs

The installation of transverse rumble strips (TRS) is a common practice to reduce vehicle speed and alert drivers on the roadway. However, the combination of TRS with road studs to enhance traffic safety is still not widely used by local authorities. This paper aims to evaluate the effectiveness of and the public perception about the installation of TRS with road studs. A field observation was carried out on the TRS road section with and without road studs for speed evaluation. A questionnaire survey was also conducted to assess the public perception towards the installation of this measure. The results indicate that the mean speed at the TRS road section with road studs was lower than the speed at the TRS road section without a road stud and its effectiveness was evaluated using a statistical test. Responses from the questionnaire survey showed that respondents agree that both TRS with and without road studs can reduce speed effectively. Respondents also believed that the installation of road studs might cause damage to the vehicle. This paper concludes that TRS installed with road studs effectively reduces speed which is consistent with the results of the public perception survey

Assessing the Reach, Adoption, Implementation, and Maintenance of the Systems Analysis and Improvement Approach for prevention of mother-to-child transmission of HIV in Manica Province, Mozambique 2018-2021 (the SAIA-SCALE Program)

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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 minutes 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

Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)—where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs