El-Sharif Hazim F, Phan Quan T, Reddy Subrayal M
Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Anal Chim Acta. 2014 Jan 27;809:155-61. doi: 10.1016/j.aca.2013.11.052. Epub 2013 Dec 5.
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.
我们研究了基于丙烯酰胺的牛血红蛋白（BHb）、马肌红蛋白（EMb）和牛过氧化氢酶（BCat）分子印迹聚合物（MIPs）在制备、洗涤和再加载阶段的缓冲溶液组成和pH值的影响。我们研究了水、磷酸盐缓冲盐水（PBS）、三（羟甲基）氨基甲烷（Tris）缓冲液和琥珀酸盐缓冲液。在整个研究中，MIP对丙烯酰胺的选择性最高，其次是N-羟甲基丙烯酰胺，然后是N-异丙基丙烯酰胺MIPs。与非印迹聚合物（NIPs）相比，MIPs的选择性根据缓冲条件和pH值的不同而降低，顺序为Tris>PBS>琥珀酸盐。Tris缓冲液在50 mM和pH 7.4时提供了最佳的印迹条件，聚丙烯酰胺中印迹BHb的MIP选择性从最初的8:1增加到128:1。值得注意的是，再加载阶段的缓冲条件对于确定MIP选择性很重要，而制备阶段的缓冲条件则不太关键。我们证明，一旦使用Tris或PBS缓冲液（pH7.4）对MIPs进行调节，应避免在水中重新加载蛋白质，因为对MIP印迹能力的负面影响会导致低选择性，仅为0.8:1。此外，将缓冲溶液的pH值酸化至低于5.9也会对MIP选择性产生负面影响，尤其是对于具有高静电荷点的蛋白质。这些缓冲调节效应在实际生物样品（即血浆和血清）的MIP效率方面也得到了成功证明。
