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

AUTOLAB APPLICATION NOTESurface Plasmon Resonance withthe Autolab SPR instrumentsIntroductionSurface Plasmon Resonance is a surfacesensitive optical technique that can be used tostudy nano thin (organic) layers on noble metalfilms. The technique is based on measuring thechange in refractive index due to, for examplebinding of an organic layer on a metal surface.TheoryThe surface plasmon resonance effect is basedon the interaction between electromagneticwaves of incident light and free electrons in aconducting surface layer. When light is reflectedat the interface of two dielectric media, totalinternal reflection will take place above the socalledcritical angle. At the same time, the lightwill generate an evanescent field. Thiselectromagnetic field has maximum intensity atthe surface of the dielectricum. In case a metallayer is present in the evanescent field, the fieldcan be enhanced and the free electrons on theouter surface (also called: surface plasmons)can resonate with the field. This is called surfaceplasmon resonance. In cases where thisresonance effect takes place, light will betransferred to surface plasmon waves and onewill thus see a decrease in the amount ofreflected light. The angle at which this decreaseoccurs is called the resonance angle, see figure1. Because the wave vector of light in air isalways smaller than the wave vector in the noblemetal surface, aFigure 1: Schematic view of SPR principle.laser beamsampleϕ SPRevanescent fieldgold layerdetectorglassimmersion oilhemi-cylindrical lensangleBare gold disk“dip”prism is needed between the sample and air tomake sure that the wave vectors in both mediacan be the same. The SPR effect is usefulbecause any change in the dielectric constant(or the refractive index) will result in a change inthe resonance angle.MethodsThere are a few different ways to measure theresonance angle. Most well known are themethods:1. Used by Biacore in which a convergent lightbeam is used. This beam results in a number ofincidence and reflecting angles. The resonanceangle then shows up on the detecting photodiodeas a dark spot. Advantage of this methodis that no mechanical parts are needed to controlthe angle of incidence. Disadvantage is that theresolution in refractive index is usually onlyaround 10 -3 .2. Used in the Autolab SPR instrument (seefigure 2) in which the reflection is measured as afunction of the incident angle of the light beam.The incident angle is changed by using avibrating mirror. The advantage of this system isthat within a short time a broad range of incidentangle can be measured. In the vibrating mirrorset-up, the angular shift is measured for a noncoatedgold sensor surface with a resolution ofapproximately 0.05 millidegrees, correspondingto a refractive index resolution of approximately1*10 -6 . For a coated gold sensor surface, theangular shift is measured with a resolution ofapproximately 0.1 millidegrees.APPLICATION NOTE 10 AUTOLAB INSTRUMENTS ARE DEVELOPED AND PRODUCED BY METROHM AUTOLAB B.V. IN THE NETHERLANDS WWW.METROHM-AUTOLAB.COM

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

AUTOLAB APPLICATION NOTEAnother option is the use of a so-called sensorchip(Pharmacia AB, Sweden) which is a wafercovered with a dextran coated gold-layer. Thedextran coating is very suitable to studymacromolecular interactions. Three methods areused to couple ligands:Immobilization of the ligand. The ligand iscoupled covalent either by amine functionalgroups or by thiol functional groups to thedextran layer.Non-covalent binding of biotinylated ligand to astreptavidin-immobilized sensor chip. Due to thesevere biotin-streptavidin interaction, it ispossible to regenerate the surface withoutbreaking the non-covalent biotinstreptavidinbond. This method is suitable to couple syntheticDNA molecules to the surface.Immobilization of capturing antibodies. Capturingantibodies are used when the activity ofantibodies is reduced by the immobilizationprocedure. Detection of antigens is achieved inthree steps. Firstly, immobilization of thecapturing antibody (for example anti-Rabbit AntiMouse-Fc). Secondly, binding of the secondantibody (a mouse antibody) by the capturingantibody. Thirdly, specific binding of the antigenby the second antibody. Regeneration of thesurface will usually break all non-covalentinteractions.These methods have been used to studybiomolecular interactions intensively. Examplesof biomolecular interactions are:peptide-antibody interactionprotein-antibody interaction, epitope mappingprotein-DNA interactionprotein-polysaccharide interactionprotein-virus interactionprotein-cell interactionprotein-T cell receptor interactionantibody-antibody interaction, capturing antibodyDNA-DNA interactionElectrochemical Surface Plasmon Resonance(ESPR)Through the combination with an AutolabPotentiostat, the Autolab SPR instrument is verywell suited for ESPR experiments, where SPRphenomena are studied in-situ during anelectrochemical experiment. This combination oftechniques has been described by a number ofdifferent groups for example to study thechanges in a thin organic film upon change ofthe electrostatic field (or potential of theelectrode). This technique is thus well suited forbiosensor development, the development ofmodified electrodes, electropolymerization, etc.Experimental exampleIn the figure below a cyclic voltammogram isshown of toluidine blue in 0.1 M KCl solution. Theexperiment was performed in the cuvet of anAutolab SPR instrument, where the gold layer ofthe glass was used as working electrode. A Ptwire was used as counter electrode and aminiature Ag/AgCl electrode as a reference. Thechange in the SPR resonance angle (or therefractive index) indicating the adsorption of theToluidine molecule in the oxidized state is shownin the blue curve. It is clearly visible that uponoxidation of the toluidine molecule adsorption onthe surface takes place (positive change in SPRangle). In the reduced state, the toluidinemolecule is back in solution again.APPLICATION NOTE 10 AUTOLAB INSTRUMENTS ARE DEVELOPED AND PRODUCED BY METROHM AUTOLAB B.V. IN THE NETHERLANDS WWW.METROHM-AUTOLAB.COM