We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K_D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

中文翻译：

---

用于选择性蛋白质识别的肽表位印迹聚合物微阵列。SARS-CoV-2 RBD蛋白的应用

我们介绍了一种实用的通用方法，用于生成基于表位印迹的聚合物微阵列，用于表面等离子共振成像（SPRi）芯片上的蛋白质识别。SPRi 平台允许以多重且无标记的方式快速筛选靶标结合动力学。通过开发用于识别 SARS-CoV-2 刺突蛋白受体结合域 (RBD) 的高度亲和力分子印迹聚合物 (MIP)，证明了此类微阵列作为合成和筛选平台的多功能性。将来自 RBD 的特征性九肽 GFNCYFPLQ 和其他对照肽微点到金 SPRi 芯片上，然后电合成多聚东来素纳米膜，一步生成 MIP 阵列。对基本合成参数（包括模板肽的表面密度及其序列）进行单芯片筛选，得到的 MIP 的解离常数 ( K _D ) 在 RBD 较低的纳摩尔范围内，超过了 RBD 对其天然靶标血管紧张素的亲和力-转化酶2酶。值得注意的是，相同的 MIP 以更高的亲和力结合 SARS-CoV-2 病毒样颗粒，并且对甲型流感 (H3N2) 病毒具有出色的辨别力。虽然用截短的七肽模板GFNCYFP制备的MIP仅表现出对RBD的亲和力略有降低，但模板氨基酸序列中的单个错配，即用丝氨酸取代中央半胱氨酸，完全抑制了RBD结合。