DEVELOPMENT OF A MICROFLUIDIC BIOSENSOR FOR THE DIAGNOSTICS AND TYPING OF MYCOBACTERIUM TUBERCULOSIS

Abstract

Background: Despite the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with this disease spread in the Russian Federation remains ex-tremely tense. At the same time, the diagnosis is performed according to the standard scheme, which takes about a month; another month is spent on the drug sensitivity tests. Thus, the development of new methods for the diagnostics and typing of mycobacteria, as well as their practical implementation is an urgent problem. The modern approaches in the field of microfluidic technologies open great opportunities in this direction. Aims: Development of a method for identification and typing of My-cobacterium tuberculosis using a label-free biosensor based on surface waves in a one-dimensional photonic crystal (PC SM biosensor). Methods: Oligonucleotide probes were selected and synthesized as DNA targets for M. tuberculosis typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, Leuconostoc mesenteroides dextrans and bovine serum albumin. The experiments were carried out using a PC SM biosensor. Results: The sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis using the PC SM biosensor. Their 3'-ends were modified in order to create extended single-stranded regions which are not subjected to the formation of secondary structures and facilitate hybridization with a sin-gle-stranded DNA target. Several series of experimental PC surface modifications were carried out using L. mesenteroides dextrans with different functional groups (including real-time detection of the modification results). The simultaneous registration of the increment layer size and volume refrac-tive index of the mixture excludes the use of a reference cell. Besides, the experiments were carried out to detect the specific binding of biotinylated oligonucleotide probes to the modified PC surface. Conclusions: A technique for the design of probes has been developed and a model system of olig-onucleotides for the detection of single-stranded DNA using a PC biosensor has been proposed. The developed technique for the PC surface modification with dextrans from L. mesenteroides allows increasing the sensitivity of oligonucleotides detection using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including those used for the identification of resistance markers.