Abstract: 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 x 10-16 to 2.5 x 10-6 mol/L in acetonitrile with the lowest determining concentration of 2.5 x 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%.
Template and target information: testosterone
Author keywords: surface plasmon resonance, Molecularly imprinted film, testosterone, surface grafting