Figure 1.
Kretschmann configuration and key parameters obtained from the full SPR angular spectra.
A) A simplified chart of the Kretschmann configuration enabling plasmon excitations and SPR measurements. The intensity of the reflected light from a monochromatic light source is measured as a function of incident light angle (θ). The light passes from a high refractive index medium (glass, ε0) to a low refractive index medium (air or liquid, ε1+εbulk). In between, the light is reflected from an interface containing a metal with a high density of free electrons and an optimal thickness for plasmon excitation (gold 50 nm, ε2) to a photodetector. The surface plasmons on the metal surface are excited at a certain incident light angle (θ) and the evanescent field created by the plasmon extends to the adjacent low refractive index medium (ε1) where samples are introduced to the system. B) A schematic full SPR angular spectrum showing the positions of the TIR region, the main SPR peak angular position and the main SPR peak minimum intensity.
Figure 2.
MDCKII cell morphology and viability on polystyrene and gold coated SPR sensor slide.
Light microscopy images of MDCKII cells cultured on A) polystyrene with a cell seeding density of 1×105 cells/cm2, B) SPR sensor slide with a cell seeding density of 5×104 cells/cm2 , C) SPR sensor slide with a cell seeding density of 7×104 cells/cm2 , D) SPR sensor slide with a cell seeding density of 1×105 cells/cm2, E) SPR sensor slide with a cell seeding density of 7×104 cells/cm2 after exposing the cell monolayer to increasing concentration (2.5 μM, 25 µM and 250 µM) of propranolol during 1 hour at a flow rate of 10 μl/min in the SPR flow channel, F) cell viability of MDCKII cells grown on polystyrene reference and gold coated SPR sensor slides. The cell seeding time used for the cell monolayers in A)–F) was 3 days. The scale bar in all images is 100 µm.
Figure 3.
Simulated full SPR spectra for optical changes within the evanescent field and thick sample layers.
Behavior of simulated full SPR angular spectra when A) changing the real and B) changing the imaginary parts of the refractive index components, and C) for very thick (waveguide) sample layers. The following parameters were used for simulations: A) sample layer thickness: 10 nm, k = 0 and n varied from 1.45–1.5, B) sample layer thickness: 10 nm, n = 1.45 and k varied from 0.00–0.05, and C) n = 1.38, k = 0 and sample layer thickness varied from 400–700 nm.
Figure 4.
Simulated full SPR spectra for optical changes within different regions of a cell monolayer.
A) Schematic representation of the sample layer used in simulating full SPR angular spectra of cell monolayers. The cell monolayer was theoretically split into three sections in order to clarify the effect of changing different optical properties in it: 1) a thin section in the magnitude of evanescent field close to the sensor surface (ñef, def = 500 nm), 2) a thick section consisting of the rest of the cell (ñcell, dcell = 3000 nm), and 3) an infinite bulk medium layer (ñbulk, dbulk = ∞). Simulated full SPR angular spectra when changing; B) the real (nef) and C) the imaginary (kef) parts of the refractive index for a cell monolayer within the evanescent field, D) the real part of the refractive index (ncell) for a cell monolayer not within the evanescent field and E) the imaginary part of the refractive index (kcell) for a cell monolayer not within the evanescent field. Insets in B-E are more detailed views of the TIR regions. The following parameters were used for simulations: B) dcell = 3000 nm, kcell = 0.002, ncell varied from 1.340–1.345, def = 500 nm, nef = 1.34 and kef = 0.002, C) dcell = 3000 nm with ncell = 1.340, kcell varied from 0–0.005, def = 500 nm, nef = 1.34 and kef = 0.002, D) dcell = 3000 nm, kcell = 0.002, ncell = 1.34, def = 500 nm, kef = 0.002 and nef varied from 1.340–1.345, and E) dcell = 3000 nm, kcell = 0.002, ncell = 1.34, def = 500 nm, nef = 1.34 and kef varied from 0–0.005.
Figure 5.
SPR signal responses during drug stimulation of MDCKII cells.
A) Measured full SPR angular spectra of a pure gold coated SPR sensor slide (grey line) and MDCKII cell monolayer immobilized on the SPR sensor slide (black line). B) Measured changes in the angular position of the SPR peak minimum as a function of time when a MDCKII cell monolayer was stimulated with propranolol (blue line) and D-mannitol (red line). C) Focused part of full SPR angular spectra showing the main SPR peak curves measured before (black line), during (red line) and after (blue line) stimulating a MDCKII cell monolayer with 25 µM propranolol. D) Measured changes in the SPR peak minimum intensity as a function of time when a MDCKII cell monolayer was stimulated with propranolol (blue line) and D-mannitol (red line). In figure C) and D) the downward arrows represent the time of sample injections, and upwards arrows represent the injection of buffer without sample.
Figure 6.
Change in SPR peak angular position versus minimum intensity during drug stimulation of MDCKII cells.
Individual repetitions of the SPR measurements in a concentration series from three repetitions when MDCKII cell monolayers were stimulated with propranolol and D-mannitol, respectively: 2.5 nM (A: propranolol, F: D-mannitol), 250 nM (B: propranolol, G: D-mannitol), 2.5 μM (C: propranolol, H: D-mannitol), 25 μM (D: propranolol, I: D-mannitol) and 250 μM (E: propranolol, J: D-mannitol).