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Peculiarities of Surface Plasmon Resonance Method Application for the Investigation of Biomolecules and Biomolecular Interactions


Biosensing is a growing field with main applications for registration and analysis of (bio)molecular interactions in biochemical, biomedical, pharmaceutical, diagnostic, and environmental industries [1, 2]. Biosensors based on surface plasmon resonance (SPR) as representatives ofsurface optical affinity biosensors possess several unique features, among which are label-free detection, realtime operation, and high sensitivity [3]. SPR and localized SPR (LSPR) biosensors have been applied for characterization of various (bio) molecular systems, including small molecules [4, 5], nucleotides [6, 7], viruses [8,9], peptides [10,11], and proteins [12,13]. Substantial scientific efforts are concentrated on developing novel sensor elements and improving the sensitivity of SPR and LSPR biosensors [14,15].

This chapter presents the results of specific practical studies of biomolecular interactions with SPR and LSPR techniques. The first study deals with SPR measurement of IgG-anti-IgG immunological reaction and calculation of surface molecular concentration and

Molecular Plasmonics: Theory and Applications Volodymyr I. Chegel and Andrii M. Lopatynskyi Copyright © 2021 Jenny Stanford Publishing Pte. Ltd.

ISBN 978-981-4800-65-5 (Hardcover), 978-0-429-29511-9 (eBook) mass of respective biomolecular coverings. The second study is focused on developing an LSPR sensor based on gold nanoparticle arrays fabricated using nanoimprint lithography and reports sensor capability to monitor antigen-antibody specific reactions between bovine serum albumin (BSA) and anti-BSA immunoglobulin.

Experimental Procedure of Surface Plasmon Resonance Technique

The SPR detection of biomolecules or their interactions with quantitative determination of mass of the substance (that is present at the sensitive surface of the sensing instrument) involves the following operations:

• The sensitive surface of a metal (gold, silver) layer of the sensor (Fig. 4.1) is brought into contact with an ambience (say, air or water) whose refractive index is known.

Simplified scheme of SPR sensor

Figure 4.1 Simplified scheme of SPR sensor.

  • • The angular dependence of the intensity of reflected light is measured over the range of angles of incidence 6>min < в < втах (here 0min and 6max are the minimal and maximal angles, respectively; they are chosen in such a way that the above range includes the plasmon resonance angle).
  • • The optical parameters (the refractive index n + ik and thickness d of conducting film) of the layered structure in the absence of the substance to be analyzed are determined from the results of measurements using the procedure of fitting the experimental and theoretical SPR curves. Usually, the parameters n + ik and d of the conducting film are known.
  • • The conducting film surface is brought into contact with a sample of the substance to be analyzed in such away that the latter could be adsorbed or, in some other way, be bound to the surface.
  • • The angular dependence of the intensity of reflected light is measured.
  • • The refractive index of the adsorbed substance nx is set. It may be chosen in different ways, so that the condition nx > n0 (where n0 is the refractive index of the ambience) is fulfilled.

The optimal upvalue is that close to the refractive index of the substance to be analyzed. For instance, if the substance to be analyzed is protein molecules, and their study is performed in a water solution, then one may take nx = 1.45.

  • • The thickness dx of the layer of the substance to be analyzed is determined from the results of measurements using the procedure of fitting the experimental and theoretical SPR curves.
  • • Knowing the optical parameters n and d of the layer studied, one can determine the mass of the adsorbed substance using the following equation obtained from the expression for molecular refraction of the substance analyzed [16]:

where Г [g/cm2] is the mass of the adsorbed substance; d(n) is the thickness (refractive index) of the layer studied; and n0 is the refractive index of the ambience. The typical value of dn/dc (i.e., the ratio between the refractive index and solution concentration increments) for different proteins is 0.188 cm3/g [17]. It should be noted that the only true physical quantity that is invariant under variation of n and d values and characterizes the actual amount of adsorbed substance is the rvalue, while nx and dx are not physically meaningful at the above measurement procedure. If the optical characteristics of a multilayer sensor structure vary with time, then the angular position of SPR varies accordingly. To register the kinetics of variation of the angular position of SPR, one should measure both the total SPR curve and the angular position of SPR at set intervals. In such way, one can register the kinetics of additional mass (say, biomolecules) inflow to the area of damping electric field of surface plasmon, as well as kinetics of mass outflow from that area. Then the kinetic curves 0sp(t) (SPR sensograms) can be analyzed using special-purpose computer software to obtain the kinetic constants of association, dissociation, etc. If biomolecules have expressed eccentricity (like immunoglobulin, etc.), the method of SPR response estimation (Chapter 2, Section 2.2.2) could be used to improve the accuracy of measurements, as shown below in the original experimental research [18].

Surface Plasmon Resonance Study of IgG-Anti-IgG Biospecific Reaction

Biospecific reactions were investigated between the complementary (biospecific) pairs of IgG-anti-IgG immunoglobulins and between protein G and IgG in the flow mode using phosphate-buffered saline (PBS) as a buffer solution (pH 7.3). Concentrations of both IgG and anti-IgG solutions were 200 pg/mL. Glycine buffer solution (pH 2.2) was used to evaluate the reaction specificity. Experimental kinetic dependences of the SPR angular position for IgG-anti-IgG and protein G-IgG specific reactions are presented in Figs. 4.2a and 4.2b.

Immunoglobulins are described as molecules with an expressed eccentricity (size 3 nm x 4 nm x 25 nm) [19], so the theoretical approach described in Subsection can be applied to this biostructure. As mentioned above, the theoretical approximation presented relates to monolayers. From this point of view, parts A-B and A-C of the immunological response (Fig. 4.2a) can be theoretically considered as monolayers with different shape parameters ^ for IgG molecules and IgG-anti-IgG complexes. Here f = h^/hL (where Лц and h± are the semiaxes of ellipsoid, parallel (| |) and perpendicular (_L) to the substrate surface plane), which defines the shape of the molecules of similar mass (see Subsection

(a) Kinetic dependence of the SPR angular position for the IgG- anti-IgG reaction

Figure 4.2 (a) Kinetic dependence of the SPR angular position for the IgG- anti-IgG reaction. The right axis reflects the surface concentration of the IgG monolayer (to level B) or the IgG-anti-IgG complex (to level C); <56 and 6C are deviations of the SPR angular position (directions of deviations are indicated by arrows); level D is associated with the specificity of reaction (if difference between levels В and D is close to zero, specificity is close to 100%). (b) Typical comparative kinetic dependence of the SPR angular position upon adsorption of IgG molecules on the bare gold surface (dashed line) and the surface covered with protein G molecules (solid line): A is the SPR response for adsorption of IgG onto the bare gold surface after washing with PBS; В is the same response for a surface modified by protein G; C is the difference associated with the modification of the surface of the sensor. Reprinted from Ref. [18], Copyright 2008, with permission from Elsevier.

The SPR angular position after rinsing with low-pH glycine buffer indicates that the effect of non-specific adsorption of anti-IgG on the SPR response in this case is small and the gradient between levels В and C reflects preferably the binding of anti-IgG molecules to specific sites of IgG molecules and complex formation. The value of £ for the IgG-anti-IgG complex can be considered to be much smaller than the value for one IgG molecule due to the increased eccentricity of the complex relative to one molecule and, accordingly, the shift in the SPR angular position after IgG-anti-IgG binding has to be bigger. This assumption correlates with the calculated values of the SPR shift (see Fig. 2.11a) and with values observed in Fig. 4.2a for kinetic dependence, where the SPR angular position for the binding of anti-IgG is significantly greater than that for the adsorption of the IgG monolayer despite the same concentration of IgG and anti- IgG (200 pg/mL) and approximately identical IgG and anti-IgG molecular weights. It is worth noting that the result shown in Fig. 4.2a represents the maximum reported response to the anti-IgG binding, since when the experiments were repeated, there was a deviation in the SPR response within certain limits, denoted as 6B and SC. This strong deviation is often observed in SPR experiments; it can be easily explained by the change in the parameter <( for the monolayers under consideration during IgG adsorption onto the gold surface and for the IgG-anti-IgG binding process. This assumption is confirmed by SPR experiments with the oriented immobilization of IgG molecules using protein G (see Fig. 4.2b). It is known that the protein G molecules, immobilized as the first layer, promote the position of immunoglobulin normally oriented relative to the surface of sensor [20] (see Fig. 4.3a).

(a) Scheme of possible orientation of IgG molecules relative to the gold surface modified by protein G

Figure 4.3 (a) Scheme of possible orientation of IgG molecules relative to the gold surface modified by protein G. (b) Scheme of possible orientation of the IgG-anti-IgG complex relative to the gold surface. Here £a > > £c- Reprinted

from Ref. [18], Copyright 2008, with permission from Elsevier.

The sequence of comparative SPR experiments shows a clear difference in the SPR response for IgG immobilization onto the gold surface that was initially modified by protein G. The rational explanation for the observation of this difference lies in the presence of an initial layer of protein G, which may affect the normal orientation of IgG molecules relative to the gold surface. This explanation is acceptable, since SPR response after the removal of specifically immobilized IgG molecules (using glycine buffer, pH 2.2) is the same as the response to the randomly adsorbed IgG monolayer on the bare surface of gold, so the SPR response value in this case depends on the part of the normally oriented IgG molecules for both comparative experiments.

Following the theoretical approach (Chapter 2, Section 2.2.2) for the results presented here, the SPR response in this study depends on the spatial position of the individual biomolecule or biomolecular complex on the Au surface, if the shape of these objects is elongated, as in the case of immunoglobulin. Clearly, the difference in the orientation of such biomolecules is not the same as the difference between prolate and oblate ellipsoids; rather, it is a strict approximation. However, there is a high probability that a maximum SPR response will be observed when individual molecules or complexes are oriented perpendicular to the gold surface when immobilized (position c in Fig. 4.3b). For the intermediate position b, the response decreases and becomes minimal when the complex or molecule is oriented parallel to the surface (position a). In addition, it can be assumed that the presence of molecular complexes of type c at sufficiently high concentrations provides a clear SPR angle shift since for the positions a and b, which are simulated by oblate ellipsoids, the SPR angle shift weakly depends on the shape of the particle, as the calculations showed.

The surface roughness of Au is important in the disordered nature of the IgG adsorption and increases the values of SB and SC.

The influence of the shape of biomolecules on the deviation of the SPR response becomes smaller with decreasing eccentricity of the biomolecule shape and is minimal for spherical objects.

The above-mentioned consideration of the spatial shape of biomolecules makes it possible to calculate nomograms for monolayers of a certain type of biomolecules or biomolecular complexes, taking into account their geometric shape (or position). Based on the assumption that the monolayers of biomolecules in the described experiments are located on a surface with average distances between molecules in the order of their linear size, nomograms were calculated for determining the surface concentration of IgG molecules or IgG-anti-IgG complexes (see Fig. 4.4).

Calculated dependences of the angular SPR response on the surface concentration of IgG molecules and IgG-anti-IgG complexes

Figure 4.4 Calculated dependences of the angular SPR response on the surface concentration of IgG molecules and IgG-anti-IgG complexes. For positions 1 and 3, ( = 0.15; for positions 2 and 4, £ = 2. Reprinted from Ref. [18], Copyright 2008, with permission from Elsevier.

The deviation of the observed surface concentration Ns (shaded area) is due to the value of the parameter £ which describes the spatial position of the IgG molecule or the IgG-anti-IgG complex. It is worth to note that nomograms consider only specifically constructed complexes, without taking into account the possible non-specific adsorption of anti-IgG on the gold surface. Thus, with the help of the SPR method, an approximate estimate of molecular adsorption can be made directly in units of the surface concentration Ns, which in the experiments under consideration was about (1.1 ± 0.1) x 1012 cm"2. In addition, Eq. (4.2) can be also used to calculate the biomolecular layer mass in mg/m2 [21]:

where Mis the molecular weight of the biomolecule or biomolecular complex and Лт = (x± + X)fi (where the indices _!_ and || indicate that the molecules are perpendicular or parallel to the surface) considering that the number of prolate and oblate particles is the same. The mass of the IgG layer was determined as equal to 3.66 ±

0.5 mg/m2, while the mass of the complex layer was 6.5 ±1.0 mg/m2, which is approximately twice as much, taking into account the observed weak non-specific adsorption.

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