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Modification of the immunecensor sureace for the antibacterial antibodies immobilisation

Previous studies had shown the effectiveness of immune based SPR biosensor for recording specific antibody-antigen interactions, and proved the possibility of using it for Rapid Determination of P. aeruginosa and S. typhimurium. For this transducer immobilized on the surface of antibacterial antibodies. Immobilization of antibodies on the gold surface provided selectively binding to an antigen of bacterial culture.

It turned out that the analysis essentially depends on how prepared the gold layer on the glass surface of the manner in which this surface is pre-treated and the state of immobilized biological material on the surface transducer.

Drawing on different coatings of golden coat can increase the sensitivity immunosensor. Specifically, the touch surface modification by dodecanoyl increases it 1.3 times. In addition, if the immobilization of biological molecules regular physical adsorption on a clean surface gold or pretreated dodecanoyl or polyelectrolyte pretty much of it can be blocked due to contact active sites (e.g. antigen-binding sites) on the surface. To avoid this shortcoming, using various approaches, including the most common are oriented inclusion of biological molecules in the tissue Langmuir-Blodgett different composition or previous immobilization on a surface protein A from Staphylococcus aureus and lectins with affinity to the Fc fragment of IgG or its carbohydrate component. In our studies, special attention is paid to optimizing the conditions selective immobilization of biological material.

This article presents experimental data for comparative use of polyelectrolyte, thiols and dextran derivatives for the immobilization of antibacterial antibodies to ensure process efficiency and operational stability by preparing surface of transducer.

Transducer’s surface modification of thiols has little effect on the sensitivity analysis and determining the intensity of its interaction with specific immune antibodies contained in serum is approximately the same as without the use of thiols.

This is due to the fact that the orientation of antibody determinants on the gold surface transducer not conducive to their maximum exposure as in the case when applied pre-polyelectrolyte layer. However, such a modification stabilizes the adsorption of antigen and enhancing the reproducibility of test results. The discrepancy results when measuring one sample is much smaller compared to the case where the antibodies are immobilized directly on the surface of gold. Thus, it is shown that the use dodecanoyl and PAA polyelectrolyte coating layer of gold on the surface of the converter allows primarily to increase the reproducibility of sensing elements of the immune optical biosensor based on outage.

It is shown that the preparation of the sensitive surface thus significantly increases the sensitivity analysis, which in this case was 10 cells /ml, and the linear segment plot of SPR signal on the number of bacterial culture in the solution is in the range of concentrations from 10 cells /ml to 106 cells /ml. Sensitivity immunosensor using antibody monolayer formed on the gold surface without modifications was 100 cells / ml, linear plot graphics in the range of 10-100 cells /ml. It was established that the formation on the surface of the transducer multilayer (three-dimensional) branched structure of antibodies using polyelectrolyte and glutaraldehyde can significantly increase the sensitivity of SPR imunosensora.

The effectiveness of selective immobilization of biological material on the surface of the transformer outage, previously covered with different chemical agents (dodecanoyl, polyelectrolyte), to create a unified functionally stable As sensitivity and specificity of the biosensor SPR is high enough for express analysis of samples for the purpose of rapid indication of bacteria then there is no need to use high-value compounds and complicate the analysis algorithm. Use polyelectrolyte for surface modification of immune converter SPR biosensor based on the most appropriate, affordable and simple.

It should also be added that the total duration of the analysis developed immunosensor is just over 40 minutes, including the time spent on the immobilization of antibodies on the surface transducer, blocking free binding sites and washing the measuring cell. The very same test procedure does not exceed 10 minutes, and it does express analysis, especially considering the fact that the surface transducer equipped with interchangeable plates, which can be pre-prepared and used as needed.

SPR analysis may be carried out in the field, as part of the instrument for its implementation can be portable form. All this creates conditions for a simple, fast and cheap approaches to control of spread of pathogenic bacteria.

Key words: bacteria, P. aeruginosa, S. typhimurium, immunosensor, transducer, polyelectrolyte, biochips, surface plasmon resonance.

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