Core–Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum,ACS Applied Materials & Interfaces

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Core–Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2017-07-17 00:00:00 , DOI: 10.1021/acsami.7b05844
Jingjing Xu ₁ , Karsten Haupt ₁ , Bernadette Tse Sum Bui ₁

Affiliation

We describe the application of a fluorescently labeled water-soluble core–shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. p-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core–MIP was first synthesized, using a thermoresponsive polymer based on N-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core–MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core–shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications.

中文翻译：

核壳分子印迹聚合物纳米颗粒作为夹心荧光免疫测定人血清中胰蛋白酶的合成抗体

我们描述了荧光标记的水溶性核-壳分子印迹聚合物（MIP）在荧光免疫分析（FIA）中检测胰蛋白酶的应用。对氨基苯甲inhibitor（PAB），一种胰蛋白酶的竞争性抑制剂，被固定在微量滴定板的孔中，从而能够将胰蛋白酶捕获在定向位置，从而保持其天然构象。选择性结合胰蛋白酶的荧光MIP纳米颗粒用于定量。MIP是通过多步固相合成方法在用PAB功能化的玻璃珠上制备的，以相同的方式定向所有胰蛋白酶分子。核心–MIP首先使用基于N的热敏聚合物合成-异丙基丙烯酰胺，以使其能够通过简单的温度变化而从固定的模板轻松释放。该壳主要由烯丙基胺组成，用于引入伯氨基以进行荧光素异硫氰酸酯（FITC）的后缀合，该壳原位嫁接在核心MIP上，其结合腔仍被固定的胰蛋白酶结合并保护。最终的核-壳MIP被赋予了均一的高亲和力结合位点，并且都具有相同的方向。MIP与其他丝氨酸蛋白酶和无关蛋白没有交叉反应或几乎没有交叉反应。我们基于MIP的FIA系统已成功应用于检测加标到未稀释的人血清中的低胰蛋白酶，其定量下限为50 pM，

更新日期：2017-07-18

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