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.
The development of hydrogel-based molecularly imprinted polymer (HydroMIPs) technology for the memory imprinting of proteins and for protein biosensor development presents many possibilities, including uses in bio-sample clean-up or selective extraction, replacement of biological antibodies in immunoassays and biosensors for medicine and the environment. Biosensors for proteins and viruses are currently expensive to develop because they require the use of expensive antibodies. Because of their biomimicry capabilities (and their potential to act as synthetic antibodies), HydroMIPs potentially offer a route to the development of new low-cost biosensors. Herein, a metal ion-mediated imprinting approach was employed to metal-code our hydrogel-based MIPs for the selective recognition of bovine serum albumin (BSA). Specifically, Co(II)-complex based MIPs exhibited a 66% enhancement (in comparison to our normal MIPs) exhibiting 92 ± 1% specific binding with Q values of 5.7 ± 0.45 mg BSA/g polymer and imprinting factors (IF) of 14.8 ± 1.9 (MIP/ non-imprinted (NIP) control). The proposed metal-coded MIPs for protein recognition are intended to lead to unprecedented improvement in MIP selectivity and for future biosensor development that rely on an electrochemical redox processes.
14We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-
Background 12The determination of drugs, metabolites and biomarkers in biological samples 13 continues to present one of the most difficult challenges to analytical scientists. 14 Matrices such as plasma, serum, blood, urine or tissues for example, usually 15 contain the analyte(s) of interest at low concentration in the presence of many 16 other components which may interfere directly or indirectly with the accurate 17 determination of species and concentration. Historically, the most common 18 methods have involved some form of extraction or isolation such as liquid-liquid 19 extraction (LLE), solid phase extraction (SPE) or protein precipitation. For a 20 recent review of sample preparation methods for bioanalysis, see [1]. This 21 includes comments on costs, automation, and miniaturisation with an overall 22 focus on productivity. 23 24Accurate quantitative measurement over the last 40 years has traditionally been 25 carried out by chromatography, mainly high performance liquid chromatography 26 (HPLC) and occasionally gas chromatography (GC). Although a range of 27 detectors has been available for both, most typically, HPLC used ultraviolet (UV) 28 and GC used flame ionisation and then both have used mass spectrometry (MS). 29Sample preparation has been usually by a variant of LLE, SPE or protein 30 precipitation [2]. As the need for greater sensitivity has been a constant 31 challenge, sophisticated and more selective methods of sample preparation have 32 been explored. One of the most attractive of these has been the use of 33 immobilised antibodies [3] selectively bind the analyte, in a similar way to an antibody. Immobilized 57 antibodies can be very specific but they are inherently quite fragile molecules, 58 particularly when exposed to organic solvents, pH values of more than 2-3 units 59 from neutral and/or heat. They can also be quite time-consuming to produce, in 60 many cases requiring repeated dosing to animals, with no certainty that useful 61 antibodies will eventually be obtained. In contrast, MIPs are produced rapidly in 62 the chemistry laboratory and use well-established synthetic routes which lead to 63 comparatively lower production costs. They are more stable over a wider pH 64 range and can be used with a broader range of solvents. This potentially also 65 offers the advantage that they could be re-usable, further lowering the costs. solid phase microextraction (SPME) or ultrasonic assisted SPME [17][18][19] and 80 matrix dispersant SPME [20, 21]. MIPs which are integrated with magnetic 81 nanoparticles offers the added advantage of a simple separation using a magnet 82 following the selective template (analyte) binding/extraction step. Ding et al. 2014 83 [22] has written a recent review on surface imprinting technologies for nano-84MIPs. This described both small and large molecule templates in two different 85 sections. Examples of biomacromolecules that have been imprinted include 86 lysozyme, bovine haemoglobin, human haemoglobin, amylase and bovine serum 87 albumin (BSA) as...
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 min 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.
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