Istituto di Chimica del Riconoscimento Molecolare, CNR , Via Mario Bianco 9, 20131 Milano, Italy.
Istituto per lo Studio delle Macromolecole, CNR , Via Corti 12, 20133 Milano, Italy.
Langmuir. 2016 Oct 11;32(40):10284-10295. doi: 10.1021/acs.langmuir.6b02816. Epub 2016 Sep 29.
In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide "click" reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation.
在本文中，我们报告了一种聚合物的后聚合修饰（PPM），以引入新的功能，从而实现用于微阵列应用的点击化学反应。母体聚合物命名为共聚（DMA-NAS-MAPS），由 N,N-二甲基丙烯酰胺（DMA）组成，DMA 通过氢键或范德华力等弱相互作用自吸附到不同的材料上；3-（三甲氧基硅基）丙基甲基丙烯酸酯（MAPS）通过与待涂覆表面的硅氧烷基团形成共价键，增强涂层的稳定性；N-丙烯酰氧基琥珀酰亚胺（NAS）是一种活性酯基团，对亲核试剂具有很高的反应活性，能够实现生物探针的固定化。这种共聚物已被广泛用于微阵列应用的表面涂层，但由于活性酯（NHS 酯）的潜在水解，存在一些局限性。NHS 酯的降解会限制这种涂层在某些情况下的使用，例如在探针不能通过微点样情况固定化，但在大体积的情况下，例如在微通道衍生化或微/纳米粒子功能化中。为了克服 NHS 酯的局限性，我们开发了一系列聚合物，它们源自常见的共聚前体，通过将聚合物链中含有的活性酯与双官能胺反应。特别是，通过 PPM 在聚合物中引入的官能团，可以实现点击化学反应，如叠氮化物/炔烃或硫醇/马来酰亚胺“点击”反应，与适当修饰的生物分子发生反应。这些反应的优点是产率定量、官能团正交性以及反应对 pH 值不敏感。新的点击官能团以定量产率插入，提高了涂层的稳定性，能够直接从溶液中固定生物分子，避免减少体积（pL）的探针点样。最后，我们证明了点击表面在高效固相 PCR 中用于 G12D KRAS 突变基因分型的适用性。
