Post-Imprinting-Modified Molecularly Imprinted Nanocavities with Two Synergetic, Orthogonal, Glycoprotein-Binding Sites to Transduce Binding Events into Fluorescence Changes
Post-Imprinting-Modified Molecularly Imprinted Nanocavities with Two Synergetic, Orthogonal, Glycoprotein-Binding Sites to Transduce Binding Events into Fluorescence Changes
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DOI：
10.1002/cnma.201800519
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发表时间：
2019-02
期刊：
影响因子：
3.8
通讯作者：
Takahiro Morishige;Eri Takano;Hirobumi Sunayama;Yukiya Kitayama;T. Takeuchi
中科院分区：
文献类型：
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作者：
Takahiro Morishige;Eri Takano;Hirobumi Sunayama;Yukiya Kitayama;T. Takeuchi
我们开发了敏感和选择性的聚合物纳米腔糖蛋白检测使用一种新的分子印迹技术，涉及后印迹修饰（PIM）。它允许在每个纳米腔中引入两个不同的相互作用位点和荧光报告分子。使用肝细胞癌生物标志物甲胎蛋白（AFP）作为模型糖蛋白。对于基于PIM的分子印迹，制备通过二硫键与可聚合基团缀合的AFP，并通过AFP聚糖之间的环状二酯形成以定向固定化方式将其固定在4-羧基-3-氟苯基硼酸固定化的基底上，促进均匀AFP印迹纳米腔的产生。用官能单体、共聚单体和交联剂进行表面引发的受控/活性自由基聚合。裂解二硫键和环状二酯以产生AFP印迹的纳米腔，产生仅存在于纳米腔内部的游离巯基。通过腔内PIM将硫醇反应性荧光染料添加到纳米腔中，产生能够将AFP结合事件转换成荧光变化的信号AFP印迹纳米腔。参考蛋白显示出很小的响应，而AFP的检测限为0.27 ng/mL（约1.5 ng/mL）。 3.9 pM）。 因此，所提出的基于腔内PIM的分子印迹技术对于用于糖蛋白的ELISA相关的无抗体传感系统是有效的。
We developed sensitive and selective polymeric nanocavities for glycoprotein detection using a novel molecular imprinting technique involving post‐imprinting modification (PIM). It allowed the introduction of two different interaction sites and a fluorescent reporter within each nanocavity. The hepatocellular carcinoma biomarker α‐fetoprotein (AFP) was used as a model glycoprotein. For PIM‐based molecular imprinting, AFP conjugated with polymerizable groups by disulfide bonding was prepared and immobilized on a 4‐carboxy‐3‐fluorophenylboronic acid‐immobilized substrate by cyclic diester formation between AFP glycans in an oriented immobilization manner, facilitating the creation of homogeneous AFP‐imprinted nanocavities. Surface‐initiated controlled/living radical polymerization was performed with a functional monomer, co‐monomer, and crosslinker. The disulfide bonds and cyclic diesters were cleaved to create AFP‐imprinted nanocavities, generating free thiol groups present inside the nanocavities only. A thiol‐reactive fluorescent dye was added to the nanocavities by in‐cavity PIM, yielding signaling AFP‐imprinted nanocavities capable of transducing AFP‐binding events into fluorescence changes. Reference proteins showed little response, while the limit of detection of AFP was 0.27 ng/mL (ca. 3.9 pM) in diluted human serum. Thus, the proposed in‐cavity PIM‐based molecular imprinting technique is effective for ELISA‐relevant, antibody‐free sensing systems for glycoproteins.