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Study of the interaction of the bioactive compounds by SPR imaging system

Final Activity Report Summary - SIBAC-SPR (Study of the interaction of the bioactive compounds by SPR imaging system)

The main goal of the project was construction of a home-made facility which makes available the Surface plasmon resonance (SPR) imaging investigations in the University of Bialystok. SPR spectroscopy has been widely and successfully used for obtaining information on a variety of molecular interactions. In general, one type of molecule - the probe - is immobilised onto the SPR sensor surface and the molecule of interest - the analyte - is brought into close contact, either using a flow-through system or a reaction chamber. As the SPR sensor is highly sensitive to changes in the mass bound to the surface, the interaction of the molecules can be monitored. The constructed SPRI facility will enable to broaden investigation potential of the host institution.

The first stage of the project was purchasing of the SPRI facility elements: laser, CCD camera and elements of the optical system (prism, lenses, pinholes, polariser etc). The SPRI facility was then constructed and adjusted. Model experiments with the immobilisation of well investigated avidine were performed in order to find the best glass type for the sensor and the best supplier for gold covered glasses.

The SPRI mapping technique is used for the investigation of interactions between biomolecules such as proteins, phospholipids, cholesterol and DNA. In this project, the home-made facility constructed in the first stage of the project was used for the investigation of interactions between proteins: cystatin C and papain as well as cystatin C and cathepsin B.

Cystatin C is commonly present in body fluids where it protects tissues from attack by bacteria proteases. Very high cystatin C concentration is indicated in cerebrospinal fluid. It is supposed that cystatin C is a 'body-guard' of this fluid. Cystatin C forms stable complexes with proteases such as cathepsins, as well as papain. Papain is an enzym-protein of plant origin. It splits peptide bonds and in this manner hydrolyses other proteins.

Cathepsins are animal lisosomal proteinases. Cathepsins play a variety of roles, such as protein digestion, limited proteolysis of biologically active proteins (including enzymes) and hormones. A range of cathepsins is present in living organisms. Different cathepsins differ slightly in their secondary structure. Cathepsins actively participate in the process of tumor invasion, especially in the methastasis phase. Therefore, cathepisins belong to tumour markers. During tumour disease, cathepsins pass into blood, urine and cerebrospinal fluid. Therefore, the cathepsin determination in body fluids is significant in tumor diagnosis.

During the project, the new SPRI sensor was developed which responds to cathepsins and papain. The sensor itself is a small glass platelet covered with a 50 nm gold layer. To this gold surface, cystatin C was fixed using a surface chemistry approach. Cystatin C 'catches' cathepsin or papain from solution. This effect is recorded by SPRI measurement.

During the period of the project, several different kinds of glass were tested along with different kinds of gold layer. The best kind of glass was selected as well as the best method for gold layer thickness.

The next step was the selection of the method for cystatin C immobilisation. Several kinds of linking thiols were tested as an intermediate monolayer between gold and cystatin C.

The constructed SPRI facility was applied for the investigation of protein-protein interactions. Such interactions enable the qualitative determination of the presence of an analyte. The interactions of immobilised cystatin C with cathepsin B, as well as cystatin C with papain create very strong signals. Therefore, the developed sensor is highly sensitive. The newly developed sensor was optimised in terms of pH, time of reaction and cystatin C concentration as well as tested for papain or cathepsin B concentration. The analysed concentration of the analytes were of the order of 10e-4-10e-5 microgram/ml i.e. significantly lower than in the case of currently used methods. Thus, the sensor for cathepsin B or papain has been developed. The sensor has the potential to be used in tumour diagnosis.