The paper describes the rational design and truncation of high-affinity DNA aptamer ApTnI12 targeting cardiac troponin I (cTnI), a key biomarker for early diagnosis of cardiac injury. By combining molecular modeling, surface plasmon resonance, and bioluminescent assay, a series of truncated variants of ApTnI12 were obtained and studied to elucidate the role of its structural elements in mediating the aptamer's affinity and specificity. The G-quadruplex motif was shown to be essential for maintaining the aptamer stability and to play a key role in the cTnI epitope recognition, during which the flanking duplex regions and loops significantly contributed to binding strength and specificity through electrostatic and hydrogen bonding interactions. The specificity of the aptamer consisting of only G-quadruplex was significantly reduced, highlighting the need for careful monitoring of its non-specific binding. A rationally designed random duplex aptamer variant achieved comparable affinity to SELEX-derived aptamers, though with a compromise in specificity. These findings emphasize the importance of balancing structural elements in the aptamer design and underscore the need for integrated computational and empirical approaches for developing aptamers with optimal performance in biosensing and therapeutic applications.

A highly sensitive solid-phase microplate immunoassay for urine survivin detection was designed based on NLuc luciferase, the bioluminescence reporter. The approach was tested on model and clinical human urine samples with the results correlating well with those obtained by a commercial colorimetric method.

Miniature electronic sensors manufactured using modern silicon technology have been intensively studied as candidates for replacing chemical and biological test systems used in medicine for precision detection of proteins and molecules in liquids and gases. Selective recognition of low concentrations of biomarkers will make it possible to diagnose dangerous diseases at early stages, thereby ensuring their successful treatment. In this study, a method for electrical detection of a heart-type fatty acid-binding protein (hFABP) in air is proposed. To enhance the selectivity, silicon nanowire field-effect transistors (Si-NW FETs) with channel widths of 0.4, 1, and 3 mu m have been fabricated and pre-functionalized with an anti-hFABP DNA aptamer (FABPAp1c-t38). It has been found that the FABPAp1c-t38 and hFABP protein induce opposite shifts of threshold voltage Vth of the Si-NW FET. For a detected target, the voltage Vth shifts from +0.2 to +2.8 V. It has been established that the voltage Vth is a better signal as compared with other electrical characteristics of the transistor. This has allowed the hFABP detection at concentrations of 1 pM in a model buffer system. It is expected that the proposed cardio target sensors and method for detection under dry conditions will contribute to the development and production of various electronic devices for application in medicine and other fields.

Tick-borne encephalitis virus (TBEV), a highly pathogenic infectious agent that causes serious damage to the nervous system is mainly transmitted by Ixodidae ticks. The laboratory methods (immunoassay and the PCR-based one) are successfully used to detect the virus in tick samples thereby avoiding unwarranted immunoprophylaxis. However, there is a need to determine the tick infection outside the laboratory conditions. In this study, we have developed a one-stage (of mix-and-read type) method for detecting virus in biological samples based on split NanoLuc complementation assay. Artificial NanoLuc luciferase split fragments NLuc(N-residue), 17.6 kDa, and NLuc(C-residue), 11 a.a., were genetically fused with the protein prED3 (fragment of the TBEV capsid protein E) or mouse anti-TBEV single-chain antibody 14D5a in all possible variants. The corresponding hybrid proteins were synthesized in E. coli recombinant cells, purified and studied. Assembling of the luciferase fragments into a bioluminescent complex proceeded due to antigen–antibody affinity interaction. The most efficient luciferase complementation was observed for the pair 14D5a-NLucCter + prED3-NLucNter: the integral bioluminescence of the complex was 2.4

Exosomes are a type of membrane vesicle secreted into the extracellular medium by most cell types. They have a great potential for clinical practice as noninvasive biomarkers for diagnosis of various diseases, prognosis, and monitoring of therapy, which stimulates the development of simple methods for isolating exosomes from biological fluids. A novel affine material based on aminosilanized superparamagnetic core‒shell nanoparticles for fast isolation of urinary exosomes is reported. Iron oxide nanoparticles coated with amino organosilane have been synthesized. The structural and magnetic characteristics of the resulting nanoparticles have been studied by transmission electron microscopy and ferromagnetic resonance. The surface of the synthesized nanoparticles has been chemically functionalized with lectin (concanavalin A), and the efficiency of the obtained material as a sorbent for affine exosome isolation from human urine has been demonstrated. A highly purified fraction of exosomes 90-200 nm in size has been obtained. The exosomal nature of the isolated vesicles has been confirmed by bioluminescent solid-phase microassay of tetrasporine receptor markers. The presence of exosomal miR-21 in the isolated human urine exosome samples has been established.

Magnetic iron oxide nanoparticles modified with aminosiloxane ( y-Fe2O3@SiO2-NH2) were used as a carrier to select DNA aptamers for a small protein tumor biomarker survivin (BIRC5, 16.5 kDa) from a randomized synthetic DNA library by the SELEX method. Survivin was covalently immobilized on the nanoparticles' surface. The library enrichment was monitored by solid-phase bioluminescent immunoassay and the DNA melting method. The prospects are outlined for the use of the obtained nanoparticles for the effective selection of aptamers specific to a given target.

Melanoma inhibitory activity protein (MIA) does obviously offer the potential to reveal clinical manifestations of melanoma. Despite a pressing need for effective diagnosis of this highly fatal disease, there are no clinically approved MIA detection ELISA kits available. A recommended MIA threshold has not yet been defined, mostly by reason of variability in immunoglobulins' affinity and stability, the difference in sample preparation and assay conditions. Here we present a pair of high-affinity DNA aptamers developed as an alternative recognition and binding element for MIA detection. Their stability and reproducible synthesis are expected to ensure this analysis under standard conditions. The devised aptamer-based solid-phase microassay of model standard and control human sera involves luciferase NLuc as a highly sensitive reporter. Bioluminescence dependence on MIA concentration ranges in a linear manner from 2.5 to 250 ng mL-1, providing a MIA detection limit of 1.67 ± 0.57 ng mL-1.

This review presents the data on the structure of bioluminescent systems of marine animals that use coelenterazine-dependent luciferases as light-emitting proteins. It is shown that these luciferases and their genetic variants with new useful properties are successfully used as reporter molecules in a variety of analytical systems in vitro and in vivo due to the availability, stability, and high quantum yield of the reactions. Their application ensures a high level of sensitivity, simple design, and fast analysis.

A biosensor based on field-effect transistors on silicon-on-insulator structures (SOI-biosensor) is a high-potential device for detection of biological molecules, for instance, such as troponin I; the biosensor allows conducting label-free real-time analysis.The aim of the study is the development of SOI-biosensor design for detection of acute myocardial infarction marker -troponin I.A notable feature of this design was the integration of two grounding electrodes directly onto the biosensor surface, which effectively nullified the static potential of the liquid sample and minimized physical breakdowns of biosensor elements.Materials and Methods.The highly specific anti-troponin I DNA aptamer was used as a receptor for specific detection of protein marker.Aptamer immobilization on the biosensor surface was carried out by physical adsorption.The analyzed range of target troponin I molecules concentration in the sample varied within 10 -11 to 10 -9 mol/L, mirroring clinical levels observed in myocardial infarction cases.During the experiment, a constant voltage of V ds =0.15 V was maintained.Results.The developed SOI-biosensor successfully detected target troponin I molecules at a concentration of 10 -11 mol/L.The detection process exhibited an effective time of approximately 200-300 s per sample.Moreover, analysis of the detection process revealed a noticeable decrease in current within the source-drain circuit, indicative of the negatively charged complex formed by troponin I and anti-troponin I DNA-aptamer at the "liquid sample-nanowire" phase interface.

NanoLuc (NLuc) is an artificial coelenterazine-dependent luciferase generated from the deep-sea shrimp Oplophorus gracilirostris. Its peculiar properties─small size and long-lasting bright bioluminescence triggered with the synthetic substrate furimazine─have made this enzyme popular as a reporter in a variety of analytical systems. Basically, to ensure the assay specificity, NLuc is genetically fused to the polypeptide with affinity for the corresponding target. The approach, however, has a limitation for non-protein biospecific molecules, thus forcing the production of biospecific luciferase derivatives via chemical conjugation. Unfortunately, it yields a heterogeneous product and often results in the loss of a significant part of bioluminescence activity. Here, we report on NLuc site-directed conjugation by combining these two approaches: several luciferase derivatives, genetically extended with hexapeptides carrying a unique Cys residue, were obtained, and the variant with activity equal to that of the intact NLuc was found. Biospecific molecules of the most commonly used types (low-weight hapten, oligonucleotide, antibody, and DNA aptamer) were chemically attached to this NLuc variant through the unique Cys using an orthogonal conjugation approach. The resulting conjugates were tested as labels in the bioluminescence assay and were shown to detect the corresponding molecular targets (e.g., cardiac markers) with high sensitivity.

Цель исследования. Оценить содержание сурвивина в моче как диагностического маркера рака мочевого пузыря (РМП); изучить прогностическое значение полиморфизма -31G>C (rs9904341) в промоторной области гена BIRC5 в отношении агрессивности течения РМП среди населения Красноярского края. Материалы и методы. Иммуноанализом определен сурвивин в 43 образцах мочи пациентов: с РМП — 27, другим вариантом злокачественного новообразования — 4, воспалительными заболеваниями мочеполовой системы и доброкачественной гиперплазией — 8, здоровых — 4. Разработанным авторами способом на основе биолюминесцентного анализа генотипированы образцы ДНК 285 пациентов с РМП и 183 здоровых доноров. Количественные данные сравнивали U-тестом Манна-Уитни, критерием χ2 Пирсона — частоты генотипов среди случаев РМП и контролей. Ассоциацию между полиморфизмом и РМП оценивали по отношению шансов с 95 % доверительным интервалом, p < 0,05 считали значимым. Результаты. Установлено, что определение сурвивина в моче позволяет разделять пациентов с РМП и здоровых с чувствительностью 66,7 % и специфичностью 100 %. Повышенное содержание сурвивина обнаружено в образцах пациентов с воспалительными заболеваниями и доброкачественными гиперплазиями мочевыводящих путей. Показано, что полиморфизм -31G>C (rs9904341) для пациентов Красноярского края при оценке риска возникновения РМП и развития рецидива заболевания не является значимым. Носительство аллеля GG является возможным предиктором агрессивного течения заболевания с быстрым прорастанием в мышечную стенку мочевого пузыря (48,7 % vs 35,7 %, p = 0,02). Заключение. Сурвивин является хорошим преддиагностическим маркером для выявления пациентов с заболеваниями мочевых путей: его повышенный уровень в моче может указывать на развитие злокачественных (в т. ч. РМП) и доброкачественных гиперплазий, а также воспалительных заболеваний уротелия. Разработка отечественной «аларм» тест-системы по определению сурвивина в моче для быстрой и неинвазивной диагностики перспективна. Полиморфизм -31G>C (rs9904341) можно рассматривать как предиктор агрессивного течения РМП.

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering "character".

Цель исследования. Оценить содержание сурвивина в моче как диагностического маркера рака мочевого пузыря (РМП); изучить прогностическое значение полиморфизма -31G>C (rs9904341) в промоторной области гена BIRC5 в отношении агрессивности течения РМП среди населения Красноярского края. Материалы и методы. Иммуноанализом определен сурвивин в 43 образцах мочи пациентов: с РМП — 27, другим вариантом злокачественного новообразования — 4, воспалительными заболеваниями мочеполовой системы и доброкачественной гиперплазией — 8, здоровых — 4. Разработанным авторами способом на основе биолюминесцентного анализа генотипированы образцы ДНК 285 пациентов с РМП и 183 здоровых доноров. Количественные данные сравнивали U-тестом Манна-Уитни, критерием χ2 Пирсона — частоты генотипов среди случаев РМП и контролей. Ассоциацию между полиморфизмом и РМП оценивали по отношению шансов с 95 % доверительным интервалом, p < 0,05 считали значимым. Результаты. Установлено, что определение сурвивина в моче позволяет разделять пациентов с РМП и здоровых с чувствительностью 66,7 % и специфичностью 100 %. Повышенное содержание сурвивина обнаружено в образцах пациентов с воспалительными заболеваниями и доброкачественными гиперплазиями мочевыводящих путей. Показано, что полиморфизм -31G>C (rs9904341) для пациентов Красноярского края при оценке риска возникновения РМП и развития рецидива заболевания не является значимым. Носительство аллеля GG является возможным предиктором агрессивного течения заболевания с быстрым прорастанием в мышечную стенку мочевого пузыря (48,7 % vs 35,7 %, p = 0,02). Заключение. Сурвивин является хорошим преддиагностическим маркером для выявления пациентов с заболеваниями мочевых путей: его повышенный уровень в моче может указывать на развитие злокачественных (в т. ч. РМП) и доброкачественных гиперплазий, а также воспалительных заболеваний уротелия. Разработка отечественной «аларм» тест-системы по определению сурвивина в моче для быстрой и неинвазивной диагностики перспективна. Полиморфизм -31G>C (rs9904341) можно рассматривать как предиктор агрессивного течения РМП.

The paper presents results on preparation and testing the properties of microsphere functional materials derived from coal fly ash ferrospheres and cenospheres for application in biotechnology and medicine including (i) magnetic affine sorbents with the functionalized surface being chemically active in biospecific binding for isolation of recombinant proteins from biological liquids, and (ii) precursors of 90Y/177Lu-aluminosilicate microspheres as sources of β-irradiation for brachytherapy.

Purpose : To study the relationship between the -31G/C (rs9904341) polymorphism in the promoter region of the survivin protein gene and the predisposition to bladder cancer (BC) in patients of the Krasnoyarsk region. Material and methods . The allelic composition of the studied gene was determined in a group of 158 BC patients, consisting of 30 women and 128 men (mean age 65.6 ± 10.7, median: 66.5; C 25 –C 75 : 59–72). The control group included 117 healthy donors and consisted of 27 women and 90 men with an average age of 60.2 ± 5.1 (median: 60; C 25 –C 75 : 57–63.25). The allelic composition was determined using the bioluminescent method. A sample with the GC genotype confirmed by sanger sequencing (center for collective use “genomika”, Novosibirsk, Russia) was used as a control. The Mann–Whitney U test was used to compare quantitative data. the studied sample was in Hardy–Weinberg equilibrium (p>0.5). The pearson χ 2 test was used to compare the frequencies of gene variants among BC cases and control samples. The association between variants rs9904341 and BC was assessed in terms of odds ratio (OR) with a 95 % confidence interval (CI); p values<0.05 were considered significant. Results . The allelic composition was determined for the genes of patients and control group participants: GG – 62 (39.2%) vs 43 (36.8%); GC – 82 (51.9%) vs 54 (46.2%); CC – 14 (8.9%) vs 20 (17.15%). The relationship between the presence of the C allele and BC was assessed using the recessive inheritance model, combining all carriers – heterozygotes and homozygotes. The frequency of occurrence of genotypes for patients and the control group was established: GG + GC – 144 (91.1%) vs 97 (82.9%); CC – 14 (8.9%) vs 20 (17.1%). Thus, carriers of the CС genotype were significantly less in patients: OR (95% CI) 0.47 (0.23–0.98), p=0.04. The relationship with tumor invasion was not significant (p=0.08). Conclusion . Based on the results of detecting the rs9904341 (G/C) polymorphism among BC patients of the Krasnoyarsk region, a protective effect of the carriage of the CC genotype was found. In order to study the allelic composition with the threat of recurrence of the disease, additional research is needed.

Purpose: To study the relationship between the -31G/C (rs9904341) polymorphism in the promoter region of the survivin protein gene and the predisposition to bladder cancer (BC) in patients of the Krasnoyarsk region. Material and methods. The allelic composition of the studied gene was determined in a group of 158 BC patients, consisting of 30 women and 128 men (mean age 65.6 ± 10.7, median: 66.5; C25–C75: 59–72). The control group included 117 healthy donors and consisted of 27 women and 90 men with an average age of 60.2 ± 5.1 (median: 60; C25–C75: 57–63.25). The allelic composition was determined using the bioluminescent method. A sample with the GC genotype confirmed by sanger sequencing (center for collective use “genomika”, Novosibirsk, Russia) was used as a control. The Mann–Whitney U test was used to compare quantitative data. the studied sample was in Hardy–Weinberg equilibrium (p>0.5). The pearson χ2 test was used to compare the frequencies of gene variants among BC cases and control samples. The association between variants rs9904341 and BC was assessed in terms of odds ratio (OR) with a 95 % confidence interval (CI); p values<0.05 were considered significant. Results. The allelic composition was determined for the genes of patients and control group participants: GG – 62 (39.2%) vs 43 (36.8%); GC – 82 (51.9%) vs 54 (46.2%); CC – 14 (8.9%) vs 20 (17.15%). The relationship between the presence of the C allele and BC was assessed using the recessive inheritance model, combining all carriers – heterozygotes and homozygotes. The frequency of occurrence of genotypes for patients and the control group was established: GG + GC – 144 (91.1%) vs 97 (82.9%); CC – 14 (8.9%) vs 20 (17.1%). Thus, carriers of the CС genotype were significantly less in patients: OR (95% CI) 0.47 (0.23–0.98), p=0.04. The relationship with tumor invasion was not significant (p=0.08). Conclusion. Based on the results of detecting the rs9904341 (G/C) polymorphism among BC patients of the Krasnoyarsk region, a protective effect of the carriage of the CC genotype was found. In order to study the allelic composition with the threat of recurrence of the disease, additional research is needed.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100–500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.

Objective was to study single-nucleotide polymorphisms (SNP) in CAT, NCL, HSPA1L, PCDH15, and PON2 genes and their associations with hearing impairment among the people working among noise-exposed workers of the mashine-building plant (JSC «Krasmash», Krasnoyarsk, Eastern Siberia, Russia).MATERIALS AND METHODS:The 443 employees of Krasmash JSC, who have been working under conditions of increased noise for at least 1 year, were surveyed and examined. A hearing study was performed by speech and tonal audiometry. Tonal audiometry was carried out in accord with according to a standard method in the frequency range 125-8000 Hz. People with chronic hearing impairment, survivors of meningitis and family history of hearing impairment were excluded from the study. The allelic composition of the studied genes was determined in the remaining group of 288 workers (study group). Polymorphisms were detected using bioluminescent method, developed by the authors earlier. The study group comprised 122 people with hearing impairment (experimental group) and 166 people without impairment (control group).RESULTS:The genotyping results of on allelic variants rs494024 (CAT), rs7598759 (NCL), rs2227956 (HSPA1L), rs7095441 (PCDH15) and rs7785846 (PON2) showed that their frequencies in the study group did not differ and were comparable with those for the European population. No statistically significant differences were revealed in the distribution of the genotypes of the studied mutations between the experimental and control groups. Also no statistically significant associations we found between hearing impairment and availability of two or several SNPs, or these SNPs and clinical characteristics of the disease (degree of hearing impairment, tinnitus). In the group of workers with an experience of 5 to 16 years, an association was found for hearing impairment and SNP rs494024, as well as when it is combined with rs7598759.CONCLUSIONS:The associations between SNP rs7598759, rs2227956, and rs7095441 and hearing impairment were not found. In the group of workers with 5-16 year experience, this association was found for SNP rs494024, as well as when it is combined with rs7598759. Discovered associations require further study.

In this work, magnetite nanoparticles coated with polysaccharides were synthesized. Arabinogalactan and chitosan were used as polysaccharides. The possibilities of immobilization of biospecific molecules on the surface of the obtained composites were studied. Experiments on covalent immobilization of biospecific molecules on magnetic nanoparticles coated with a polysaccharide showed a high density of immobilized molecules. This suggests the use of such materials in bioanalytical systems or as affinity sorbents. (c) 2020 Elsevier B.V. All rights reserved.