Matsishin M. Development of affinity biosensor system for the detection of mutations associated with Ph'-positive leukemia and resistant forms of tuberculosis

In present work, DNA sensors for recognition of the mutations in the sequences related to the rpoB gene of Mycobacterium tuberculosis and hybrid gene bcr-abl were developed. The main goal of this research was to develop scientific and technological approaches to the development of DNA sensors for detecting mutations associated with Ph'-positive leukemia and resistant forms of tuberculosis. For detection of mutation in the codon 531 of the rpoB gene of M. tuberculosis, which causes resistance to anti-tuberculosis drug rifampicin, electrochemical (impedimetric) DNA sensor was developed. A bioselective element of the biosensor was formed on the gold electrode surface by immobilization of thiolated single-stranded DNA probes P2, which reproduce a corresponding fragment of the rpoB gene, and by passivation of the electrode surface (to reduce the non-specific adsorption) using 6-mercapto-1-hexanol. High selectivity of this biosensor has been shown: at injection of 1 nM complementary oligonucleotide T2, the charge transfer resistance (Rt) was significantly higher than that of 50 nM non-complementary oligonucleotides TC (632 Ohm and 121 Ohm, respectively). Moreover, a high level of discrimination between fully complementary oligonucleotides T2 and single-base mismatch oligonucleotides TN was achieved by the increasing hybridization stringency (reducing the ionic strength of the hybridization buffer solution). The high sensitivity, selectivity and reproducibility of the sensor response were proved in the research. For selective label-free real-time recognition of hybrid gene bcr-abl, surface plasmon resonance (SPR)- based biosensor was developed. Two stringency control strategies for detection of DNA hybridization and discrimination of fully and partially complementary 24-mer sequences were applied. These sequences are specific to the human normal bcr gene and the hybrid bcr-abl gene, protein product of which is responsible for chronic myelogenous leukemia. Thermodynamic parameters of the oligonucleotides hybridization, which were obtained by using the web server DINAMelt, allowed to suppose the possibility for these stringency control strategies based (i) on the change of the hybridization buffer ionic strength and (ii) on the temperature elevation. The first one resulted in increase of the discrimination index of completely and partially complementary oligonucleotides from 1.8 in 2xSSC to 2.9 in 0.5xSSC, for 50 nM concentration of the oligonucleotides. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~ 50 C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 C. Thus, the effective thermo discrimination of these oligonucleotides was achieved. For improvement of the sensitivity of SPR-based DNA sensor, a method of selective amplification of the sensor response using gold nanoparticles modified by oligonucleotides, which are complementary to oligonucleotides immobilized on the sensor surface was elaborated. In order to control the level of oligonucleotide immobilization on the surface of gold nanoparticles, a fluorescence-based experimental approach was proposed. The achieved selective amplification of the SPR-based DNA sensor response by modified gold nanoparticles allowed to decrease detection limit and to increase biosensor signal approximately one hundred times.