CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Portugal; BioMark/CINTESIS-ISEP, School of Engineering, Polytechnic Institute of Porto, Portugal.
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Portugal.
Anal Chim Acta. 2017 Aug 15;981:41-52. doi: 10.1016/j.aca.2017.05.017. Epub 2017 Jun 2.
An electrochemical biosensor was developed by merging the features of Molecular Imprinting technique and Screen-Printed Electrode (SPE) for the simple and fast screening of cardiac biomarker myoglobin (Myo) in point-of-care (POC). The MIP artificial receptor for Myo was prepared by electrooxidative polymerization of phenol (Ph) on a AuSPE in the presence of Myo as template molecule. The choice of the most effective protein extraction procedure from the various extraction methods tested (mildly acidic/basic solutions, pure/mixed organic solvents, solutions containing surfactants and enzymatic digestion methods), and the optimization of the thickness of the polymer film was carefully undertaken in order to improve binding characteristics of Myo to the imprinted polymer receptor and increase the sensitivity of the MIP biosensor. The film thickness was optimized by adjusting scan rate and the number of cycles during cyclic voltammetric electropolymerization of Ph. The thickness of the polyphenol nanocoating of only few nanometres (∼4.4 nm), and similar to the protein diameter, brought in significant improvements in terms of sensor sensitivity. The binding affinity of MIP receptor film was estimated by fitting the experimental data to Freundlich isotherm and a ∼8 fold increase in the binding affinity of Myo to the imprinted polymer (K = 0.119 ± 0.002 ng mL) when compared to the non-imprinted polymer (K = 0.015 ± 0.002 ng mL) which demonstrated excellent (re)binding affinity for the imprinted protein. The incubation of the Myo MIP receptor modified electrode with increasing concentration of protein (from 0.001 ng mL to 100 μg mL) resulted in a decrease of the ferro/ferricyanide redox current. LODs of 2.1 and 14 pg mL were obtained from calibration curves built in neutral buffer and diluted artificial serum, respectively, using SWV technique, enabling the detection of the protein biomarker at clinically relevant levels. The prepared MIP biosensor was applied to the determination of Myo spiked serum samples with satisfactory results.
一种电化学生物传感器是通过将分子印迹技术和丝网印刷电极（SPE）的特点结合起来而开发的，用于在即时护理（POC）中简单快速地筛选心脏生物标志物肌红蛋白（Myo）。Myo 的 MIP 人工受体是通过在 AuSPE 上电化学氧化聚合苯酚（Ph），并以 Myo 为模板分子制备的。从测试的各种提取方法（弱酸/碱性溶液、纯/混合有机溶剂、含表面活性剂的溶液和酶消化方法）中选择最有效的蛋白质提取程序，并优化聚合物膜的厚度，仔细进行，以提高 Myo 与印迹聚合物受体的结合特性，并提高 MIP 生物传感器的灵敏度。通过调整扫描速率和循环伏安聚合期间的循环次数来优化膜厚度。多酚纳米涂层的厚度仅为数纳米（约 4.4nm），与蛋白质直径相似，在传感器灵敏度方面带来了显著的改进。通过将实验数据拟合到 Freundlich 等温线，估算 MIP 受体膜的结合亲和力，并与非印迹聚合物（K=0.015±0.002ng mL）相比，Myo 与印迹聚合物的结合亲和力提高了约 8 倍（K=0.119±0.002ng mL），表明对印迹蛋白具有极好的（再）结合亲和力。用增加浓度的蛋白质（从 0.001ng mL 到 100μg mL）孵育 Myo MIP 受体修饰电极，导致铁/铁氰化物氧化还原电流减小。使用 SWV 技术，在中性缓冲液和稀释的人工血清中分别从校准曲线中获得了 2.1 和 14pg mL 的 LOD，能够在临床相关水平检测到蛋白质生物标志物。制备的 MIP 生物传感器用于测定 Myo 加标血清样品，结果令人满意。
