Journal of Clinical PracticeJournal of Clinical Practice2220-30952618-8627Eco-Vector7181510.17816/clinpract71815Research ArticleDevelopment of a microfluidic biosensor for the diagnostics and typing of <i>Mycobacterium Tuberculosis</i>MitkoTatiana V.<p>Graduate Student, laboratory assistant</p>mitko@phystech.eduhttps://orcid.org/0000-0002-0107-1906ShakurovRuslan I.<p>Junior Research Associate</p>ruslan.shakurov@rcpcm.orghttps://orcid.org/0000-0002-5986-0676ShirshikovFedor V.<p>Junior Research Associate</p>shirshikov@rcpcm.orghttps://orcid.org/0000-0001-6452-1874SvetlanaSizova V.<p>Cand. Sci. (chem.)</p>sv.sizova@gmail.comhttps://orcid.org/0000-0003-0846-4670AlievaElena V.<p>PhD</p>alieva@isan.troitsk.ruhttps://orcid.org/0000-0002-5251-7365KonopskyValery N.<p>PhD</p>konopsky@gmail.comhttps://orcid.org/0000-0001-6114-5172BasmanovDmitry V.<p>Research Associate</p>dmitry.basmanov@rcpcm.orghttps://orcid.org/0000-0001-6620-7360BespyatykhJulia A.<p>PhD</p>JuliaBes@rcpcm.orghttps://orcid.org/0000-0002-4408-503XFederal Research and Clinical Center of Physical-Chemical MedicineInstitute of Spectroscopy of the Russian Academy of Sciences2307202112214202006202123062021Copyright © 2021, Mitko T.V., Shakurov R.I., Shirshikov F.V., Svetlana S.V., Alieva E.V., Konopsky V.N., Basmanov D.V., Bespyatykh J.A.2021<p><strong>Background. </strong>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. <strong>Aim. </strong>Development of a method for identification and typing of <em>Mycobacterium tuberculosis</em> using a label-free biosensor on surface waves in a one-dimensional photonic crystal (PC SM biosensor). <strong>Methods. </strong>Oligonucleotide probes were selected and synthesized as DNA targets for <em>M. tuberculosis</em> typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, <em>Leuconostoc mesenteroides</em> dextrans and bovine serum albumin. Experiments were carried out using a PC SM biosensor. <strong>Results. </strong>Sequences of detecting oligonucleotide probes were selected for spoligotyping of <em>M. tuberculosis </em>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 <em>L. mesenteroides </em>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. <strong>Conclusions. </strong>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 <em>L. mesenteroides</em>, 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.</p>biosensortuberculosisMycobacterium tuberculosismicrofluidic technologiesbiochipsphotonic crystal surface modesphotonic crystalpersonalized medicineбиосенсортуберкулезMycobacterium tuberculosisмикрофлюидные технологиибиочипыповерхностные оптические волныфотонный кристаллперсонализированная медицина[Global tuberculosis report 2019. Geneva: World Health Organization; 2019. Available from: https://www.who.int/tb/ publications/global_report/en/][Нечаева О.Б. Состояние и перспективы противотуберкулезной службы России в период COVID-19 // Туберкулез и болезни легких. 2020. Т. 98, № 12. С. 7-19. [Nechaeva OB. State and prospects of the anti-tuberculosis service of Russia in the period of COVID-19. Tuberculosis and Lung Diseases. 2020;98(12):7-19. (In Russ).]][Global tuberculosis report 2020. Geneva: World Health Organization; 2020. Available from: https://www.who.int/tb/publica-tions/global_report/en/][Konopsky VN, Karakouz T, Alieva EV, et al. Photonic crystal biosensor based on optical surface waves. Sensors. 2013;13:2566-2578. doi: 10.3390/s130202566][Konopsky VN, Mitko TV, Aldarov KG, et al. Photonic crystal surface mode imaging for multiplexed and high-throughput label-free biosensing. Biosensors and Bioelectronics. 2020;168:112575. doi: 10.1016/j.bios.2020.112575][Bespyatykh JA, Zimenkov DV, Shitikov EA, et al. Spoligotyp-ing of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays. Infect Genet Evol. 2014;26:41-46. doi: 10.1016/j.meegid.2014.04.024][Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31(13):3406-3415. doi: 10.1093/nar/gkg595][Konopsky VN, Alieva EV. Photonic crystal surface waves for optical biosensors. Anal Chem. 2007;79:4729-4735. doi: 10.1021/ac070275y][Morozova OV, Levchenko OA, Cherpakova ZA, et al. Surface modification with polyallylamines for adhesion of biopolymers and cells. Int J Adhesion Adhesives. 2019;92:125-132][Castro-Garza J, Garsia-Jacobo P, Rivera-Morales LG, et al. Detection of anti-HspX antibodies and HspX protein in patient sera for the identification of recent latent infection by Mycobacterium tuberculosis. PLoS One. 2017;12(8):1-13. doi: 10.1371/journal.pone.0181714][Bespyatykh JA, Shitikov EA, Bespyatykh DA, et al. Metabolic changes of Mycobacterium tuberculosis during the anti-tuberculosis therapy. Pathogens. 2020;9(2):131. doi: 10.3390/pathogens9020131][Басманов Д.В., Митько Т.В., Шакуров Р.И., Беспя-тых Ю.А. Создание новых микрофлюидных биосенсоров для мультиплексной детекции процесса связывания лигандов в реальном времени // Актуальные проблемы биомедицины-2021: Материалы XXVII Всероссийской конференции молодых ученых с международным участием. Санкт-Петербург, 2021. С. 296297. [Basmanov DV, Mitko TV, Shakurov RI, Bespyatykh YuA. Creation of new microfluidic biosensors for multiplex detection of the ligand binding process in real time. In: Actual problems of biomedicine-2021: Materials of the XXVII All-Russian Conference of Young Scientists with International participation. Saint Petersburg; 2021. Р. 296-297. (In Russ).]]