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  • 1.
    Platou Vikinge, Trine
    Linköping University, Department of Physics, Measurement Technology, Biology and Chemistry. Linköping University, The Institute of Technology.
    Surface Plasmon Resonance for the Detection of Coagulation and Protein Interactions1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Medical improvements rely on methodological inventions and developments and on the steadily increasing knowledge of human physiology. This thesis presents methodological developments enabling the study of body fluid interactions with a material surface under more realistic and physiologically relevant conditions than previously possible.

    By use of the surface plasmon resonance (SPR) technique biological interactions on a sensor surface can be detected in real time as changes in refractive index. We studied biological interactions in blood-derived fluids, and experienced the difficulties related to refractive index changes from irrelevant components of the analyte interacting with the ligand on the sensor surface. However, with the use of avian antibodies, interacting minimally with the human complement system, instead of murine ones for analyte capture this problem was averted, and the human serum concentration of the C3 protein could be reproducibly determined.

    A novel use of the SPR based technique for the detection of clotting time was developed. The placement of whole blood or blood plasma samples on an SPR sensor surface gave sigmoid response signals. Curve fit analysis followed by feature extraction revealed that the SPR signal was sensitive to concentrations of coagulation activator and inhibitor in the sample. The structures left on the sensor surfaces after completed clotting (followed by a rinse and dry procedure) were imaged via atomic force microscopy (AFM). The AFM images revealed mesh-works of fibres with size distributions reflecting the concentration of coagulation activator in the sample. This supports the interpretation of the SPR signal as being due to the fibrin polymerisation process of the coagulation system.

    The detection of clotting with SPR was compared to the clinically used nephelometric technique. Statistical analysis indicated that the two methods are equally reliable, and the differences in observed clotting times could be ascribed to the difference in temperature at which the instruments were operated. Whereas nephelometric clotting detection requires plasma preparation, the SPR technique can be used also for the measurement of whole blood clotting.

    The comparison of clotting time as measured with SPR and with a quartz crystal microbalance device with energy dissipation detection (QCM-D) revealed that the former gives shorter clotting times at high concentrations of activator. This is because SPR does not require the biological material to be coupled to the surface to be detected, whereas QCM does. Without addition of coagulation activator to the sample the clotting process seemed to be surface initiated as the two methods gave literally the same clotting times. The energy dissipation detected by the QCM-D yielded interesting information on the viscoelastic properties of the clots, that could be related to the rigidity and viscosity of the samples.

    Finally, G-protein-coupled receptors were investigated, being of prime importance in the understanding of a number of presently used medical drugs, but also relevant in the understanding of platelet function and their role in the coagulation cascade. A surface specifically binding the G-protein, and not its α or βγ subunits, was constructed by immobilisation of a synthetic peptide derived from a G-protein-coupled receptor (α2A-adrenoceptor) to the SPR sensor surface. The G-protein capturing efficiency was shown to depend on the orientation and extension of the peptide on the surface, and the method is a promising tool in the study of specificity and efficiency of drugs targeted at G-proteins.

    List of papers
    1. Blood plasma coagulation studied by surface plasmon resonance
    Open this publication in new window or tab >>Blood plasma coagulation studied by surface plasmon resonance
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    1999 (English)In: BIOMEDICAL SENSORS, FIBERS, AND OPTICAL DELIVERY SYSTEMS, PROCEEDINGS, SPIE - International Society for Optical Engineering, 1999, Vol. 76, no 1, p. 107-114Conference paper, Published paper (Refereed)
    Abstract [en]

    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 mostly followed 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 (AFM) 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.

    Place, publisher, year, edition, pages
    SPIE - International Society for Optical Engineering, 1999
    Keywords
    surface plasmon resonance (SPR); blood plasma coagulation; atomic force microscopy (AFM)
    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-75805 (URN)10.1117/12.336920 (DOI)000078785700013 ()
    Conference
    Conference on Biomedical Sensors, Fibers, and Optical Delivery Systems, Stockholm, September 08-10, 1998
    Available from: 2012-03-12 Created: 2012-03-12 Last updated: 2022-06-08Bibliographically approved
    2. Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay
    Open this publication in new window or tab >>Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay
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    1999 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, E-ISSN 2567-689X, p. 917-Conference paper, Published paper (Other academic)
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-49798 (URN)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2023-08-28
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