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

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

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

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

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

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

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

Generation of ribosome imprinted polymers for sensitive detection of translational responses

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation