表面接枝法制备睾酮素分子印迹表面等离子体共振传感器

构建了一种选择性检测睾酮素的分子印迹表面等离子体共振( Surface plasmon resonance, SPR)传感器。采用紫外光引发表面接枝技术,在固定引发转移终止剂的SPR芯片表面制备了以睾酮素为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂的分子印迹膜( Molecularly imprinted film, MIF)。利用SPR在目标共振角处现场监测使MIF聚合成膜过程更易控。偏振调制-红外反射吸收光谱表征证明MIF接枝成功。原子力显微镜结果显示,MIF 表面均匀散布着纳米尺寸的孔穴。利用 SPR 对2.5×10-16~2.5×10-6 mol/L睾酮素进行吸附检测,检出限低至2.5×10-16 mol/L,对低浓度和高浓度的睾酮素吸附分段进行线性拟合,得到拟合线性方程分别为y=19.69+1.21x(R2=0.9913)和y=11.5+0.45x(R2=0.9895)；睾酮素类似物雌二醇、雌三醇和黄体酮的吸附实验结果显示,此印迹膜对模板分子有很好的选择性；5次重复洗脱吸附后,MIF仍保持较好的传感性能,说明此传感器具有较高的稳定性和重复利用性；在人工尿液样品中测得的睾酮素回收率为85.2%~92.8%,说明此传感器可以用于实际样品测定。

Molecularly Imprinted Film Grafted from Surface Plasmon Resonance Sensor Chip for Determination of Testosterone

A molecularly imprinted film ( MIF) was prepared on the surface plasmon resonance ( SPR) sensor chip for the detection of testosterone. The nanometer imprinted film was synthesized by surface grafting approach. First, the gold sensor chip was modified with an iniferter of benzyl diethyldithiocarbamate to create the “grafting from” polymeric surface. Grafting of the MIF onto the SPR chip was subsequently achieved through ultraviolet light-initiated copolymerization of methacrylic acid ( functional monomer ) and ethylene glycol dimethacrylate ( crosslinker ) in the presence of testosterone as a model template. The template molecules were then removed to form a MIF with specific recognition sites for testosterone. With this iniferter technique , self-aggregation in the reaction solution phase was avoided and grafting polymerization was confined to the exterior of the chip surface. The grafting process was implemented by in situ monitoring with SPR, which permitted the thickness of the film to be easily and strictly controlled. The chip surface modified with a testosterone-imprinted film was characterized by the methods of polarization modulation infrared reflection absorption spectroscopy ( PM-IRRAS ) and atomic force microscopy ( AFM ) . According to the results, testosterone-imprinted film modification of the SPR chip was achieved by the distribution of numerous homogeneous, nanoscale holes on the surface. The testosterone-imprinted film was applied on a SPR sensor chip for the detection of testosterone in the range of 2 . 5 í 10-16 to 2 . 5 í 10-6 mol/L in acetonitrile with the lowest determining concentration of 2. 5 í 10-16 mol/L. Adsorption experiments showed that there were two kinds of binding sites in the MIF, and the linear correlations between the changes in reflectivity and the concentration of testosterone were y=19. 69 + 1. 21x(R2=0. 9913) and y=11. 5 + 0. 45x(R2=0. 9895), respectively. Control experiments utilizing estradiol, estriol, and progesterone as analogues showed impressive selectivity and specificity for testosterone determination. The testosterone-imprinted film reproducibility was evaluated with five cycles of rinsing-rebinding and the RSD was 16 . 8% and 11 . 2% for the SPR angle changes in rinsing and rebinding respectively, demonstrating that the MIF-modified SPR sensor had good reproducibility and repeatability. Finally, the SPR sensor was successfully used to determine testosterone in artificial urine samples with recoveries from 85 . 2% to 92 . 8%.