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  • As the material for in silico experiments we used

    2021-09-14

    As the material for in silico experiments we used an amino Flutamide synthesis sequence of a fragment of HIV1 surface glycoprotein gp120 corresponding to its less mutable B-cellular epitope: NMWKNNMVEQMHEDIISLWDQ. This sequence is the same as the sequence of the NQ21 and the biotin-NQ21 peptides (the last one has a biotin molecule conjugated to its N-terminus). SERS-spectroscopy (Surface Enhanced Raman Spectroscopy) was applied to study NQ21 and biotin-NQ21 peptides (at concentration of 30 μg/mL) in water solutions with 0.01 M phosphate buffers at pH levels of 6.8, 7.4 and 8.0. In all the experiments original silver containing SERS substrates [19,20] were used with the aim to enhance the Raman signal. The SERS substrates were fabricated using a two-step process. First, porous silicon layer was formed on a highly-doped n-type silicon wafer by an electrochemical anodic etching. Second, silver nanoparticles were formed by the silver immersion deposition on porous silicon [19]. Prior to the peptides' deposition, each substrate was refreshed in diluted HCl for 30 s. Then the SERS substrates were incubated in solutions of the peptides and peptide-free buffer solution for 2 h. Before the SERS measurements we tried to register the Raman spectra of peptides deposited on the virgin porous silicon and glass slide. These experiments were performed to find if it is possible to get Raman spectra without an additional enhancement with the silvered substrates. The samples of virgin porous silicon covered with peptides were prepared in similar conditions as those of the silver-coated porous silicon. The glass slide was covered with peptides' solutions by drop deposition. The SERS-spectra were registered both for the wet and then dried at room temperature samples. SERS-spectroscopy was conducted using a 3D scanning confocal Raman microscope Confotec NR500 equipped with 633 nm laser which light passes through the 100× objective and forms a laser spot about 500 nm in diameter. This equipment provided spectral resolution about 3.5 cm−1. For the measurements peptide-coated SERS substrates were covered with a glass. Laser power after passing the objective was 0.68 ± 0.02 mW. The signal collection time was 1 s. All the spectra measured were normalized and aligned with respect to the silicon band position at 520 cm−1 that was observed due to silicon base of the SERS substrates. Native gel electrophoresis was performed for NQ21 peptide in several buffers without SDS and other denaturating agents: phosphate buffer with pH = 6.0, 7.4, 8.0; tris-glycine buffer with pH = 7.4 and 8.0; tris-barbital buffer with pH = 8.9. For the separation of proteins we used agarose gel from the Cormay gel protein 100 kit (cat. no. 6–048). Fixation, staining (amido black) and destaining solutions were from the same kit. The running time was equal to 30 min (phosphate buffers pH = 6.0, 7.4, 8.0, tris-glicyne pH = 7.4, tris-barbital pH = 8.9), or 15 min (tris-glycine pH = 8.0). The amperage was equal to 5 mA (tris-glycine pH = 7.4, pH = 8.0), 11 mA (phosphate buffers pH = 6.0, 7.4, 8.0), or 15 mA (tris-barbital pH = 8.9). We spent 1 μg of the peptide per cell. As the molecular weight markers we used a set of standards (Bio-RAD, №151–1901) that includes: horse myoglobin (17 kDa, pI = 7.2), soybean trypsin inhibitor (20 kDa, pI = 4.5), ovalbumin (44 kDa, pI = 4.5), conalbumin (76 kDa, pI = 6.3). Positions of horse myoglobin and ovalbumin were re-checked with pure horse myoglobin (Sigma-Aldridge) and pure ovalbumin (Reachim). Since the pI value for myoglobin is 7.2, it showed the shortest distance from the start point in all the experiments. Three other proteins migrated according to their molecular masses: conalbumin, ovalbumin, soybean trypsin inhibitor (in the ascending order of the migration path length). The pH influence on the fluorescence intensity of the NQ21 peptide was studied using a Solar CM2203 spectrofluorometer. The fluorescence intensity of tryptophan residues in the NQ21 peptide was measured at 350 nm (the excitation wavelength was 270 nm [21]) in seven solutions with pH varied from 5.0 to 8.5. All the solutions were phosphate buffers with the same molarity (0.01 M), but different ratios of hydrogen phosphate and dihydrogen phosphate anions. Actual pH level was measured in every solution. The NQ21 peptide concentration was equal to 1 μg/mL in all the solutions.