The absorption-based surface plasmon resonance (SPR) theory was applied to the development of a new SPR measurement method for the determination of glucose in solution, based on a color reaction. With this technique, it becomes possible to detect low molecular weight analytes by SPR, which have been regarded to be difficult to quantify, so far, by utilizing the changes of the imaginary part of the refractive index (RI) caused by structural changes of a dye molecule. In this study, the absorption maximum of the dye is coincident with the wavelength of the incident light of the SPR instrument. Under this experimental condition, no angle shift of the SPR signal is induced, but only changes of the reflectance intensity occur. Therefore, low molecular weight analytes can be determined by monitoring the reflectance intensity due to the signal amplification by the dye. As an example, the redox enzyme [glucose oxidase (GOD)] is immobilizd on a SPR sensor chip. Glucose oxidase is known to oxidize the analyte glucose accompanied by generation of hydrogen peroxide. A color reaction is observed by using the new Trinder reagent in the presence of a reductive enzyme [peroxidase (POD)], and a SPR signal broadening is induced depending on the amount of the newly formed dye. Thus, determination of the glucose concentration down to 10 nM was achieved by following this SPR signal broadening changes.
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