Influence of surface-imprinted nanoparticles on trypsin activity.
Influence of surface-imprinted nanoparticles on trypsin activity.
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DOI：
10.1002/adhm.201300634
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发表时间：
2014-09
影响因子：
10
通讯作者：
Piletsky S
中科院分区：
文献类型：
--
作者：
Guerreiro A;Poma A;Karim K;Moczko E;Takarada J;de Vargas-Sansalvador IP;Turner N;Piletska E;de Magalhães CS;Glazova N;Serkova A;Omelianova A;Piletsky S
采用固相法将模板酶（胰蛋白酶）固定在固相载体上，合成了分子印迹聚合物纳米颗粒（MIP NPs），并将其作为亲和基质用于分离高亲和性纳米颗粒组分。该方案改编自Poma et al. [1]并且允许高亲和性材料的快速合成和受控分离和纯化，一个生产周期仅持续2.5小时。以这种方式产生的材料不受模板污染，并具有亚纳摩尔的表观解离常数，同时显示出低的交叉反应性。取决于在印迹期间使用的酶固定方法（随机相对于定向，具有受保护的酶活性位点），游离酶重新结合到MIP NP上导致其抑制或表观稳定化（未观察到抑制）JIP可以被广泛地认为是天然受体/抗体的人造等同物，并且像它们的天然对应物一样能够识别和结合相应的靶分子。由于这个原因，也由于它们在诊断中取代不稳定的天然受体的潜力，MIP是一个重要的研究目标。[2-4]当以纳米颗粒形式使用时，与更“传统”的方法如研磨整料、珠粒或膜相反，MIP具有在测定、传感和亲和分离[3]和催化中用作天然受体（如抗体）的直接替代物的潜力。[5]虽然使用印迹聚合物进行的大部分工作涉及小分子，但蛋白质的印迹仍然具有挑战性[6]，因为它们的高分子量，以及大量的官能团，这些官能团在非生理条件下的低稳定性阻碍了印迹过程。这一事实，加上缺乏用于合成MIP NP的可扩展和标准方法，限制了该技术进入预期的实际和商业应用。
Molecularly Imprinted Polymer Nanoparticles (MIP NPs) were synthesized via a solidphase approach with immobilized template enzyme (trypsin) on a solid support which following polymer synthesis acted as affinity matrix for separation of high-affinity nanoparticle fractions. This protocol was adapted from Poma et al.[1] and allows for fast synthesis and controlled separation and purification of high affinity materials, with one production cycle lasting just 2.5 hours. Materials produced this way were free from template contamination and possessed sub-nanomolar apparent dissociation constants whilst showing low cross-reactivity. Depending on the enzyme immobilization method (random vs. oriented, with protected enzyme active site) used during imprinting, the rebinding of the free enzyme onto the MIP NP results either in its inhibition or in apparent stabilization (no inhibition observed).MIPs can be broadly considered as man-made equivalents of natural receptors/antibodies, and, like their natural counterparts are able to recognize and bind corresponding target molecules. For this reason, and also due to their potential to replace unstable natural receptors in diagnostics, MIPs are an important research target.[2-4] When used in nanoparticle format, as opposed to more “traditional” approaches such as ground monoliths, beads or films, MIPs have the potential to be used as direct replacement for natural receptors (such as antibodies) in assays, sensing and affinity separations [3] and catalysis.[5] While the majority of work performed with imprinted polymers deals with small molecules, imprinting of proteins remains challenging [6] due to their high molecular-weights, as well as multitude of functional groups which together with low stability in non-physiological conditions hinder the imprinting process. This fact, together with the lack of a scalable and standard process for the synthesis of MIP NPs restricts access of this technology to prospective practical and commercial applications.