We have characterized the imprinting capability of a
family of
acrylamide polymer-based molecularly imprinted polymers (MIPs) for
bovine hemoglobin (BHb) and trypsin (Tryp) using spectrophotometric
and quartz crystal microbalance (QCM) sensor techniques. Bulk gel
characterization on acrylamide (AA), N-hydroxymethylacrylamide
(NHMA), and N-isopropylacrylamide (NiPAM) gave varied
selectivities when compared with nonimprinted polymers. We have also
harnessed the ability of the MIPs to facilitate protein crystallization
as a means of evaluating their selectivity for cognate and noncognate
proteins. Crystallization trials indicated improved crystal formation
in the order NiPAM < AA < NHMA. QCM studies of thin film MIPs
confirm this trend with N-hydroxymethyl acrylamide
MIPs exhibiting best discrimination between MIP and NIP and also cognate/noncognate
protein loading. Equivalent results for acrylamide MIPs suggested
that the cavities were equally selective for both proteins, while N-isopropylacrylamide MIPs were not selective for either
cognate BHb or noncognate BSA. All BHb MIP-QCM sensors based on AA,
NHMA, or NiPAM were essentially nonresponsive to smaller, noncognate
proteins. Protein crystallization studies validated the hydrophilic
efficacy of MIPS indicated in the QCM studies.
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 μM, 44 ± 3 μM, 17 ± 2 μM 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.
We have investigated the effect of buffer solution composition and pH during the preparation, washing and re-loading phases within a family of acrylamide-based molecularly imprinted polymers (MIPs) for bovine haemoglobin (BHb), equine myoglobin (EMb) and bovine catalyse (BCat). We investigated water, phosphate buffer saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer and succinate buffer. Throughout the study MIP selectivity was highest for acrylamide, followed by N-hydroxymethylacrylamide, and then N-iso-propylacrylamide MIPs. The selectivity of the MIPs when compared with the NIPs decreased depending on the buffer conditions and pH in the order of Tris>PBS>succinate. The Tris buffer provided optimum imprinting conditions at 50 mM and pH 7.4, and MIP selectivities for the imprinting of BHb in polyacrylamide increased from an initial 8:1 to a 128:1 ratio. It was noted that the buffer conditions for the re-loading stage was important for determining MIP selectivity and the buffer conditions for the preparation stage was found to be less critical. We demonstrated that once MIPs are conditioned using Tris or PBS buffers (pH7.4) protein reloading in water should be avoided as negative effects on the MIP's imprinting capability results in low selectivities of 0.8:1. Furthermore, acidifying the pH of the buffer solution below pH 5.9 also has a negative impact on MIP selectivity especially for proteins with high isoelectric points. These buffer conditioning effects have also been successfully demonstrated in terms of MIP efficiency in real biological samples, namely plasma and serum.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.