Knut Johansen

Imaging surface plasmon resonance sensor based on multiple wavelengths: Sensitivity considerations

A new, multiple wavelength surface plasmon resonance apparatus for imaging applications is presented. It can be used for biosensing, e.g., for monitoring of chemical and biological reactions in real time with label-free molecules. A setup with a fixed incident angle in the Kretschmann configuration with gold as the supporting metal is described, both theoretically and experimentally. Simulations of the sensor response based on independently recorded optical (ellipsometric) data of gold show that the sensitivity for three-dimensional recognition layers (bulk) increases with increasing wavelength. For two-dimensional recognition layers (adlayer) maximum sensitivity is obtained within a limited wavelength range. In this situation, the rejection of bulk disturbances, e.g., emanating from temperature variations, decreases, with increasing wavelength. For imaging surface plasmon resonance the spatial resolution decreases with increasing wavelength. Hence, there is always a compromise between spatial resolution, bulk disturbance rejection, and sensitivity. Most importantly, by simultaneously using multiple wavelengths, it is possible to maintain a high sensitivity and accuracy over a large dynamic range. Furthermore, our simulations show that the sensitivity is independent of the refractive index of the prism

Total internal reflection ellipsometry: principles and applications

A concept for a measurement technique based on ellipsometry in conditions of total internal reflection is presented. When combined with surface plasmon resonance (SPR) effects, this technique becomes powerful for monitoring and analyzing adsorption and desorption on thin semitransparent metal films as well as for analyzing the semitransparent films themselves. We call this technique total internal reflection ellipsometry (TIRE). The theory of ellipsometry under total internal reflection combined with SPR is discussed for some simple cases. For more advanced cases and to prove the concept, simulations are performed with the Fresnel formalism. The use of TIRE is exemplified by applications in protein adsorption, corrosion monitoring, and adsorption from opaque liquids on metal surfaces. Simulations and experiments show greatly enhanced thin-film sensitivity compared with ordinary ellipsometry.

Surface plasmon resonance : Instrumental resolution using photo diode arrays

Surface plasmon resonance (SPR) sensors are used to study biomolecular interactions. We have performed a theoretical analysis of a SPR instrument using a convergent beam, a linear detector with various numbers of pixels and various analogue-to-digital converters (ADCs) with a corresponding resolution ranging from 8 to 16 bits. Studies of small molecules at low concentrations or with low affinities are limited by the instrumental set-up, e.g. by the resolution, linearity and noise. The amplitudes of these parameters are highly dependent on the detector, ADC and dip-finding algorithm used. We have studied several dip-finding algorithms, e.g. intensity measurements, second- and third-order polynomial fits and centroid algorithms. Each algorithm used with the ADC and the detector has a resolution associated with it. Some algorithms also have an intrinsic algorithm error that is dependent on the number of pixels and the shape of the dip. A weighted centroid algorithm that has an excellent overall performance is described. If an accuracy of 10-6 refractive index units (RIU) is satisfactory, a 12-bit ADC and a 64-pixel detector are appropriate. Theoretically, by using a 16-bit ADC and a 1024-pixel detector, a resolution of better than 10-9 RIU is obtainable.

Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay

It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.

Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance.

One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met(644)-Phe(688)) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg(645)-Arg(657) inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr(654) inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium-calmodulin signalling and for signal integration between other signalling pathways.

Sensitivity deviation: instrumental linearity errors that influence concentration analyses and kinetic evaluation of biomolecular interactions

Many scientific instruments utilise multiple element detectors, e.g. CCDs or photodiode arrays, to monitor the change in a position of an optical pattern. For example. instruments for affinity biosensing based on surface plasmon resonance (SPR) or resonant mirror are equipped with such detectors. An important and desired property of these bioanalytical instruments is that the calculation of the movement or change in shape follows the true change. This is often not the case and it may lead to linearity errors, and to sensitivity errors. The sensitivity is normally defined as the slope of the calibration curve. A new parameter is introduced to account for the linearity errors, the sensitivity deviation, defined as the deviation from the undistorted slope of the calibration curve. The linearity error and the sensitivity deviation are intimately related and the sensitivity deviation may lead to misinterpretation of kinetic data, mass transport limitations and concentration analyses. Because the linearity errors are small (e.g. 10 pg/mm2 of biomolecules on the sensor surface) with regard to the dynamic range (e.g. 30,000 pg/mm2), they can be difficult to discover. However, the linearity errors are often not negligible with regard to a typical response (e.g. 0-100 pg/mm2). and may therefore cause serious problems. A method for detecting linearity errors is outlined. Further on, this paper demonstrates how integral linearity errors of less than 1% can result in a sensitivity deviation of 10%, a value that in our opinion cannot be ignored in biospecific interaction analysis (BIA). It should also be stressed out that this phenomenon also occurs in other instruments using array detectors.

Blood plasma coagulation studied by surface plasmon resonance

A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real-time as a function of thromboplastin and heparin concentrations. The physical reason for the SPR signal observed is discussed and 3 different models are proposed. The response curves were analyzed by multivariable curve fitting followed by feature extraction. Interesting parameters of the sigmoid curves were lag time, slope and maximum response. When thromboplastin concentrations were increased, the lag-time decreased and the slope of the curve increased. A prolonged clotting time was followed mostly by increased maximum response, with exception for samples with no or very little thromboplastin added. High heparin concentrations changed the clotting kinetics. As seen from the lag-time vs. slope relation. Atomic force microscopy pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and fiber thickness increased with lower thromboplastin concentration. The results correlate well with present common methods.