A molecularly imprinted polymer (MIP) for biosensing trypsin is prepared by incorporating a benzamidine-based signaling fluorescent monomer during its synthesis. Binding to trypsin results in a 100-fold fluorescence enhancement when the MIP is excited with UV light. The assay can be performed by simply mixing a small amount of water-soluble MIP (100 μg) and trypsin together in a quartz cuvette, followed by a direct read-out on a spectrofluorimeter. There is no need to separate the free from the bound trypsin. The limit of quantification is 50 nM in phosphate buffer and 210 nM in urine. The aqueous MIP is prepared by a solid-phase synthesis method on glass-beads functionalized with a metal chelate to immobilize trypsin via its surface histidine. The active site of the enzyme is left free for binding to the benzamidine moiety of the fluorescent monomer, resulting in a sensitive MIP for probing the enzyme’s active site. Incorporation of a thermoresponsive monomer, N-isopropyacrylamide, in the polymerization mixture, yields thermoresponsive MIP nanoparticles that are released from the support by a simple temperature change, generating template-free polymers. The MIPs are endowed with improved binding site homogeneity since all binding sites have the same orientation. Their size is ∼70 nm and the dissociation constant (K_d) of the MIP-trypsin complex is 237 nM, with little or no cross-reactivity with other proteins, including serine proteases.

用于生物传感胰蛋白酶的分子印迹聚合物（MIP）是通过在合成过程中掺入基于苯甲idine的信号荧光单体而制备的。当MIP被紫外光激发时，与胰蛋白酶的结合会导致荧光增强100倍。可以通过在石英比色皿中简单地将少量水溶性MIP（100μg）和胰蛋白酶混合在一起，然后在荧光分光光度计上直接读出来进行测定。无需将游离的胰蛋白酶与结合的胰蛋白酶分开。磷酸盐缓冲液的定量极限是50 nM，尿液的定量极限是210 nM。通过固相合成法在玻璃珠上制备MIP水溶液，该玻璃珠被金属螯合物官能化以通过其表面组氨酸固定胰蛋白酶。酶的活性位点被释放以与荧光单体的苄am部分结合，从而形成了一个敏感的MIP用于探测酶的活性位点。掺入热敏性单体，在聚合混合物中，N-异丙基丙烯酰胺可产生热响应性MIP纳米粒子，该纳米粒子可通过简单的温度变化从载体中释放出来，从而生成不含模板的聚合物。由于所有结合位点具有相同的取向，因此赋予了MIP结合位点同质性。它们的大小约为70 nm，MIP-胰蛋白酶复合物的解离常数（K _d）为237 nM，与其他蛋白质（包括丝氨酸蛋白酶）的交叉反应性很小或没有。