Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning

Abstract

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.

MIP-based electrochemical protein profiling

“…Interestingly, the binding capacity is highest for BHb-MIPpolyAA while both EMb-MIPpolyAA and BCat-MIPpolyAA exhibit the lowest binding capacity. It has previously been observed that with smaller size proteins a higher crosslinking density is necessary; the opposite is also true for larger proteins [12,30]. Since the crosslinking density remained the same (10% by weight), the low MIP affinities for BCat and EMb can be attributed to the fact that fewer cavities were imprinted due too high, and too low of a crosslinking density respectively.…”

“…However, in the imprinting community, bio-macromolecules such as proteins present a variety of challenges and successful imprints are highly sought after. Proteins are relatively labile and have changeable conformations that are sensitive to various factors (e.g., solvent environments, pH and temperature) [7,[10][11][12]. Moreover, a large number of proteins are vital markers; for example, in the case of haemoglobin, mutations in genes that encode for the protein's subunits can result in hereditary diseases such as sickle cell anaemia, thalassaemia, and haemoglobinopathies [1].…”

Comparison of descriptors for predicting selectivity of protein‐imprinted polymers

“…AR and SR were estimated, as described later. MW (Pang et al ., ; Odabaşi et al ., ; Çimen et al ., ; Pan et al ., ) and IEP (Pan et al ., ; El‐Sharif et al ., ; Zhang et al ., ) have already been suggested as possible explanations for observed PIP selectivity in an ad hoc manner in the literature (Bossi et al ., ; Turner et al ., ; Bossi et al ., ; Chen et al ., ; Uysal et al ., ; Hua et al ., ; Whitcombe et al ., ; Sankarakumar and Tong, ; Li et al ., 2014), and in the succeeding texts, we formalize their effect. Functional group distribution was also suggested as a contributor to protein selectivity (Kimhi and Bianco‐Peled, ; Gai et al ., ; Levi and Srebnik, ; Zayats et al ., ; Lan et al ., ).…”

“…and IEP(Pan et al, 2013;El-Sharif et al, 2014;Zhang et al, 2014) have already been suggested as possible explanations for observed PIP selectivity in an ad hoc manner in the…”

Generation of ribosome imprinted polymers for sensitive detection of translational responses

“…MIPs found early application for solid phase extraction of drugs and pesticides 27 , and more recently for bioanalyte detection and as biosensors 28, 29 . In particular, hydrogel MIPs based on polyacrylamide (PAA) and derivatives thereof were realised for selective recognition of bio-macromolecules, including, but not limited to, small regularly shaped globular proteins such as bovine serum albumin (BSA) 30, 31 or haemoglobin 32, 33 . Protein-based MIPs were also tested as nucleates for protein crystallisation 34 .

Figure 1Schematic overview of R-MIP preparation.

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