Development of a microfluidic biosensor for the diagnostics and typing of Mycobacterium Tuberculosis

Background. Despite on the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with spreading of this disease in Russian Federation remains extremely tense. At the same time, the diagnosis is carried out according to the standard scheme, which takes about a month; another month takes test formulation for drug sensitivity. Thus, the development of new methods for diagnostics and typing of mycobacteria, as well as practice implementation of these developments is an urgent direction. Modern developments in the field of microfluidic technologies open up great opportunities in this direction. Aim. Development of a method for identification and typing of Mycobacterium tuberculosis using a label-free biosensor 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. Experiments were carried out using a PC SM biosensor. Results. Sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis on the PC SM biosensor. Modification of their 3'-ends was carried out in order to create extended single-stranded regions that are not subject to the formation of secondary structures and facilitate hybridization with a single-stranded DNA target. Several series of experimental modifications of the PC surface were carried out by using L. mesenteroides dextrans with different functional groups (including detection of the modification results real time) with simultaneous registration of the increment layer size and volume refractive index of the mixture, which excludes the use of a reference cell. Other 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 was developed and a model system of oligonucleotides for the detection of single-stranded DNA using a PC biosensor was proposed. The developed technique of modification of the PC surface with dextrans from L. mesenteroides, which allows to increase the sensitivity of detection of oligonucleotides using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including identification of resistance markers.