Surface Plasmon Resonance with the Autolab SPR ... - Metrohm

AUTOLAB APPLICATION NOTE3. Where instead of changing the angle ofincidence, the wavelength of the light ischanged. This method is used in an optical fibresetup.The SPR effect is only available on metals inwhich the electrons behave as a free electrongas, meaning that their motions are independentof the charge that they leave behind whenmoving. This limits the choice of metal to copper,silver, aluminium and gold.Figure 2: Autolab SPR optical principle.Gold is by far the most used in biologicalapplications. The thickness of the metal layerdepends very much on the optical constants ofthe bulk material as well as on the wavelength ofthe light. The Autolab SPR instrument uses 670nm as a wavelength resulting in an optimumthickness for the gold layer of about 50 nm.There is a linear relationship between the amountof bound material and shift in SPR angle. TheSPR angle shift in millidegrees is used as aresponse unit to quantify the binding ofmacromolecules to the sensor surface. Theresponse also depends on the refractive index ofthe bulk solution. A change of 120 millidegreesrepresents a change in surface protein ofapproximately 1ng/mm2, or in bulk refractiveindex of approximately 10 -3 .The detection principle limits the size of theanalyte that can be studied. If the molecularweight of the compound is below 5000 Dalton,then the change in refractive index upon bindingto the sensor surface is too low to be detecteddirectly. Only special modified layers incombination with specific small molecules willshow the direct binding. The penetration depth ofthe evanescent wave of 300-400 nm alsodetermines the size of macromolecules orparticles that can be studied. Particles largerthan 400 nm cannot be measured totally. As aresult, the signal is not linear related to theamount of bound particles. Under thesecircumstances it is possible to study the bindingqualitatively, but a quantitative or kinetic analysiscannot be performed.ApplicationsSince the SPR effect is only sensible to changesthat take place in a few hundred nm from thesurface most of the studies involve adsorbedmolecules, meaning that in cases where forexample antibody-antigen interactions arestudied, one of them will be immobilized on thesurface. With the SPR disc that comes with theAutolab SPR instrument the following options arepossible:Bare gold surfaces:Macromolecular interaction measurements canbe performed by coating the ligandelectrostatically to the surface, followed byadding the analyte. After coating, a blockingcompound is usually necessary to preventaspecific interactions. This method is especiallysuitable for detection of large particles as cellsand viruses.Biomolecular interaction measurements withbiotinylated macromolecules. Firstly, the goldsensor surface is coated with biotin, followed bybinding with streptavidin. Secondly, biotinylatedmolecules are allowed to bind with unoccupiedbinding sites of streptavidin (stoichiometrystreptavidin-biotin interaction is 1:4). Thirdly,binding of the analyte can be measured.Biomolecular interaction measurements with thiolcontaining compounds. Gold shows a stronginteraction with sulphur. By applying thisproperty for peptides, self-assembled receptorlayers are developed.Direct measurement of low molecular weightcompounds by response enhancement with latexparticles. Low molecular weight compounds canbe attached to carboxy modified latex by acarbodiimide coupling reaction Direct binding oflow molecular weight compounds coupled tolatex particles can be determined using a coatedligand at the sensor surface.Measurements on conducting polymers usingelectropolymerization.APPLICATION NOTE 10 AUTOLAB INSTRUMENTS ARE DEVELOPED AND PRODUCED BY METROHM AUTOLAB B.V. IN THE NETHERLANDS WWW.METROHM-AUTOLAB.COM