Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

Abstract

Pathogenic bacteria have become a serious socio-economic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn-proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn-proteins by superparamagnetic beads modified with chicken anti-Zn(II) antibody. S. aureus culture was treated with ZnCl(2). Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle-surface interactions were tested and associated protein profiles were identified using SDS-PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS-PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.

Remote‐controlled robotic platform ORPHEUS as a new tool for detection of bacteria in the environment

“…The detection limit of 30 bacterial cells per microliter was obtained, which is approximately 50 times higher than beads-based immunoextraction technique for S. aureus detection, but the detection limit in this work was obtained after 2 h of cultivation, while in work from Krizkova et al the enrichment cultivation was performed for 5 h [14]. Whole protocol was possible to realize by remote-controlled robot, which makes it usable for monitoring of distant areas without contamination.…”

“…The current third generation of ''smart'' robots richly equipped with sensors can be used in extreme environments (sea depths, space, volcanos). Thus, it is not surprising that the ALP is produced during the growth and sporulation of various bacterial strains (Staphylococcus aureus [14], Bacillus cereus, and Bacillus amyloliquefaciens [15][16][17], Escherichia coli [18], thermophilic bacteria [19] such as Thermotoga neapolitana, Thermus caldophilus, Thermus thermophilus, Bacillus stearothermophilus, Pyrococcus abyssi, and Deinococcus radiodurans, which is able to live in a radioactive environment [20][21][22]). Space rovers need a considerable amount of electricity to move and power their equipment, so energy-efficient scientific devices are important for remote sensing [2].…”

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide)

“…Magnetic particles were separated by a magnet and the solution was used for following measurements. Other experimental details can be found in [ 23 , 24 , 25 , 26 , 27 ].…”

Liposomal nanotransporter for targeted binding based on nucleic acid anchor system

“…Magnetic particles were separated by magnet and solution was used for measurements. Other experimental details can be found in the following papers .…”