Wavelength-scanning surface plasmon resonance imaging for label-free multiplexed protein microarray assay.

We presented wavelength-scanning surface plasmon resonance (SPR) imaging as an optical method for label-free, multiplexed assay of protein microarray. The image of reflected light was measured while the wavelength was scanned in steps at a fixed incident angle. The thickness of the gold layer deposited on the substrate was mainly ∼50 nm for collecting sample data, whereas it was ∼200 nm on a minor part for collecting reference signals. The intensity-versus-wavelength data was doubly compensated for the wavelength dependence and the temporal fluctuation. The wavelength affording minima of the reflected intensity based on SPR (SPR wavelength) were automatically determined at each pixel of the measured image. A gold-deposited substrate was firstly modified with a functional thiol and lastly immobilized with biotin. We estimated the thickness of the thiol and biotin layers as well as that of a spacer layer using our new SPR imaging. Next we monitored the specific binding of avidin to biotin immobilized on the substrate in the flow of a buffer solution. As the measurement was repeated at intervals of 10s, the SPR wavelength determined at each pixel was averaged in real-time for 20 selected areas consisting 314 pixels. The limit of detection was 20 pm in SPR wavelength corresponding to 5 pm in thickness.