Hi Ugur,

for calculating the layer thicknesses, I have made good experience with a software called "Winspall". It is free of charge and developed at the Max-Planck Institute for polymer research in Mainz (Germany). All you need is the data file of your scan measurement (of course) and the dielectric constants of the substances and materials your layers are made of.

Here is the link: http://www.mpip-mainz.mpg.de/groups/knoll/software/winspall302_free.zip

Feel free to test it. If you have any questions about the use, just ask.

http://www.mpip-mainz.mpg.de/groups/knoll/software/winspall302_free.zip

Hi Ugur,

The most simple way of calculating the adsorbed layer thickness is to compare the SPR-dip shift caused by the adsorbed layer with an experiment measuring the shift caused by known refractive index buffers e.g. glycerol. As a rule of thumb for a wave length of the light of about 700 nm the sensitivity of 1 nanogram per square mm causes a shift of 100 millidegree of the SPR dip which is related to a 1000 RU = 0.001 RIU. Start injecting 1% glycerol in water with respect to water and look which shift you will get. You will also an indication of the noise levels in terms of RU (RMSD).

Ellipsometry is very useful for such estimations, before the SPR measurements we usually are estimating thicknesses by ellisometry, than with SPR is much easier to work:

Article Study of antibody/antigen binding kinetics by total internal...

Hello Ugur,

The publication in Biomacromolecules dx.doi.org/10.1021/bm201236a gives a good account how you can calculate the thickness, if you homemade setup only produces time-sensograms. If you can also make angle-scanning data, then the Winspall method is probably better. For the first, you will need to get you instrument sensitivity (as Dr. Schasfoort described earlier). For both methods, the refractive indeces you use have a large effect on the thickness result (and also with the ellipsomtry), so please be careful with them. There are some possibilities to overcome this, like measuring in air and water or with several wavelength SPR (DOI: 10.1016/j.snb.2010.05.048).

thank you everyone for all the useful responses. I figured out the problem with this paper (jung et al "Quantitative Interpretation of the Response of Surface Plasmon Resonance Sensors to Adsorbed Films" doi: 10.1021/la971228b). it was very useful for me. Firstly I calculated the sensitivity factor of our device. then I calculated the adsorbed layer thickness by using the RIU shift and density of the analyt.

We used the formalism develoved by Jung et al. (Jung LS, Campbell CT, Chinowsky TM, Mar MN, Yee SS. Quantitativeinterpretation of the response of surface plasmon resonance sensors toadsorbed films. Langmuir 1998;14(19):5636-48.) for our home made optical fiber SPR sensor which a good agreement for a biotin/neutravidin specific interaction model (B. Sciacca, A. François, M. Klingler-Hoffmann, J. Brazzatti, M. Penno, P. Hoffmann, T. M. Monro, “Radiative-surface plasmon resonance for the detection of apolipoprotein E in medical diagnostics applications”, Nanomedicine: Nanotechnology, Biology, and Medicine 9, 550-557 (2013))

One more publication on application of ellipsomety to estimate thickness of protein layer before SPR measurements:

Article Total internal reflection ellipsometry of metal–organic comp...

Hi Ugur

What kind of layer do you have on the surface? The answers above are probably good enough for you but there is also another method based on non-interacting probes. We have used this with good results. It is described here:

Non-interacting molecules as innate structural probes in surface plasmon resonance

Langmuir 2013, Schoch et al.