A new application for x-ray lithography: Fabrication of blazed diffractive optical elements with a deep phase profile

Use of x-ray lithography to produce blazed diffractive optical elements (DOEs) is described. Proposed method allows one to make highly efficient blazed DOE with a deep phase profile (ten wavelengths and more) using a single x-ray mask with a binary transmission pattern. Unlike the well-known multilevel DOEs, blazed ones do not involve fabrication and aligning of a set of masks. DOEs with a profile depth of 10 {mu}m and more and zone sizes down to 1 {mu}m can be obtained due to the short wavelength and high penetrability of x rays. The first experimental samples of blazed DOEs with a 10 {mu}m-height profile--lenses and gratings - were fabricated by x-ray lithography with synchrotron radiation using the x-ray masks, prepared in accordance with the pulse-width modulation algorithm. Diffraction efficiency for lenses was measured for white light: it is higher than 80% for the central part of the lenses (inside a 10 mm diameter) and about 60% for an area of 20 mm diameter

Production of Bst polymerase for diagnosis of different infections using loop-mediated isothermal amplification

Introduction. The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) possesses 5'-3' DNA polymerase activity, 5'-3' displacement activity and high processivity. These properties make it possible to use Bst DNA polymerase in loop-mediated isothermal amplification (LAMP), which provides highly specific amplification of the target sequence and is used for rapid detection of agents causing human infectious diseases. The purpose of the study was to produce a recombinant Bst polymerase enzyme in the bacterial expression system and to assess its properties for LAMP-based diagnostics of infectious diseases. Materials and methods. Expression constructs carrying the Bst polymerase gene were obtained using genetic engineering techniques. Different Escherichia coli strains were used for protein expression. Metal-chelate and gel filtration chromatography techniques were used for protein purification. Catalytic characteristics of the enzyme were assessed in loop-mediated isothermal amplification reactions using AmpliSens SARS-CoV-2-IT, AmpliSens IAV-IT and AmpliSens IBV-IT diagnostic systems designed for high-quality detection of SARS-CoV-2, influenza A virus (IAV) and influenza B virus (IBV) RNA, respectively. Results. The offered protocol for production, extraction and purification of recombinant Bst polymerase makes it possible to produce the enzyme in the bacterial expression system using E. coli cells in a soluble form and reaching the yield up to 20% of the total cell mass. In LAMP reactions, the obtained enzyme demonstrates activity comparable with that of the commercial enzyme Bst 2.0 (NEB). Conclusion. Considering the fast purification and production of the enzyme, the obtained recombinant Bst polymerase can be used in LAMP-based diagnostic kits