Surface Plasmon Resonance Sensors With High–accuracy Measurement Based On Molecularly Imprinted Films Prepared By Multi–wavelength Photopolymerization

Surface plasmon resonance?SPR?is an optical phenomenon that measures changes in refractive index on the surface of metallic substrates as a result of molecular bindings or film formations.Owing to its unique advantages of rapid,label free,high–sensitivity and real time measurements,the applications of SPR have been getting more popular for medical diagnosis,food safety,and environmental monitoring.Molecularly imprinted technique is a research method of functional polymer that can bind template molecules with high selectivity and specificity.By combining the good performance of molecularly imprinted polymers?MIPs?such as high stability in severe environment,simple preparation,and high selectivity and the good performance of SPR spectroscopy,we developed molecularly imprinted SPR sensors for high accuracy detection of small molecules.Molecularly imprinted films were prepared using multi–wavelength photopolymerization initiated by ultraviolet?UV?and visible light in this thesis,which offer more choice about the MIPs preparation and SPR sensing.The main contents are as the following:1.A molecularly imprinted film?MIF?on the SPR sensor chip was prepared for the detection of estradiol by UV–initiated polymerizaiton.First,the gold sensor chip was modified with an iniferter of N,N'–diethylaminodithiocarbamoylmethylstyrene to create the“grafting from”polymeric surface.Grafting of MIF onto the SPR chip was subsequently achieved through the copolymerization of methacrylic acid?MAA,functional monomer?and ethylene glycol dimethacrylate?EGDMA,crosslinker?in the presence of estradiol as a model template.The template molecules were then removed to form MIF with specific recognition sites for estradiol.With this iniferter technique,self–aggregation in the 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 provided excellent control of the film thickness.The chip surfaces were characterized by the methods of contact angle measurements and scanning electron microscopy.A good linear relationship was obtained between the SPR angle and estradiol concentrations in the range of 1.0×10^–10 to 1.0×10^–6 mol/L in phosphate buffered saline?PBS?with the lowest determining concentration of 1.0×10^–10 mol/L.Control experiments utilizing testosterone as an analogue showed impressive selectivity and specificity for estradiol determination.Furthermore,this SPR sensor with the estradiol–imprinted film possessed high reproducibility with three sensor chips prepared independently using the same procedure.2.Two SPR sensors based on MIF using the different mass ratio of crosslinker were prepared by UV photograft polymerization for the sensitive detection of testosterone in acetonitrile and in aqueous phase respectively?template/functional monomer/crosslinker ratio of 1:4:3 in acetonitrile and 1:4:8 in aqueous phase?.The SPR sensor was characterized with the methods of polarization modulation infrared reflection absorption spectroscopy?PM–IRRAS?and atomic force microscopy?AFM?.Analysis of SPR spectroscopy reveals that the MIF showed good selectivity for testosterone compared to other analogues both in acetonitrile and aqueous phase.The detection limit was estimated to be 2.5×10^–16 mol/L in acetonitrile and 2.5×10^–12mol/L in PBS.The two sensors both can be regenerated over times without significant deterioration of the sensor performance,which shows that the MIF–based SPR sensors had high stability and reproducibility.3.A water compatible MIF was anchored onto the gold–coated glass surface using visible light–initiated photopolymerization for fabricating a SPR sensor sensitive to theophylline.The interaction between methacrylic acid and theophylline was studied by UV–Vis absorption spectrophotometric methods.Based on this study,the MIF was prepared through the copolymerization of methacrylic acid?functional monomer?and N,N'–Methylenebisacrylamide?crosslinker?in the presence of theophylline as a model template in aqueous phase using a couple of methylene blue?red–sensitized dye?and sodium p–toluenesulfinate?reducing agent?as the photo–redox initiator.The template molecules were then removed to form a molecularly imprinted film with specific recognition sites for theophylline.The SPR chip surfaces were characterized by the methods of contact angle measurements and PM–IRRAS.A good linear relationship was obtained between the SPR angle and theophylline concentrations in the range of 1.0×10^–10 to 1.0×10^–6 mol/L in PBS with the lowest determining concentration of 1.0×10^–10 mol/L.Analysis of SPR spectroscopy reveals that the MIF showed impressive selectivity and specificity for theophylline determination compared to other analogues and NIF.Furthermore,this SPR sensor with the theophylline–imprinted film possessed high reproducibility with three sensor chips prepared independently using the same procedure.This portable sensor system can be successfully applied to monitor theophylline in environmental samples.4.Considering that SPR used a laser diode??=635nm?as light source,we envisaged a method for in–situ formation of theophylline MIF on the surface of SPR device using the energy of evanescent wave field which is created by the incidence light in the condition of total internal reflection.The MIF was limited to the point of the interrogation of the following meaning binding event for the opinion of miniaturization of devices and raw materials.The MIF dot was approximately 1.5mm×1.5 mm in size and showed good recognition to template molecules in PBS.