Capillary isoelectric focusing of microorganisms in the pH range 2–5 in a dynamically modified FS capillary with UV detection

Abstract

The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2-5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10(3).

Bacterial Inactivation by Applying an Alternating Electromagnetic Field Using PAMAM Dendron-modified Magnetic Nanoparticles

“…This phenomenon was in good agreement with the surface charges of the PAMAMMNPs; i.e., PAMAM-MNPs are positively charged at pH 5.0 to 8.0, almost neutral at pH 9.0, and negatively charged at pH 10.0. 22 The surfaces of majority of bacterial cells including E. coli are negatively 23,24 From the above results, we can conclude that the binding of the PAMAM-MNPs to the cell surface was highly efficient and cell recovery was achieved, based on the electrostatic interaction between the PAMAM-MNPs and E. coli cells.…”

Testing of the influenza virus purification by CIEF

“…solution of the selected simple ampholytic electrolytes dissolved in the catholyte [45,46], HEPES (pK 1 5 3.65, pK 2 5 7.5, pI 5 5.58) and L-aspartic acid (pK 1 5 1.88, pK 2 5 3.66, pI 5 2.77) in the ratio 1:1, the segment of the sample mixture of viruses, and the segment of both the mixture of commercial carrier ampholytes and the UV detectable pI markers used for the tracing of the pH gradient in the range 2.0-7.5 or 3.0-10.0. The time of the injection of the spacer segment was 25 s, the sample segment 5-15 s and the segment of carrier ampholytes and pI markers was 40 s.…”

“…It can be assumed that the efficiency of the applied virus purification methods could be monitored also by CIEF with UV detection in an appropriately selected pH gradient [39,40,47,48]. The reproducibility and linearity of the pH gradient within pH range 2.0-7.5 were achieved by applying of the segmental injection [39,46]. In the first segment, the suitable spacers were dissolved in the catholyte solution; simultaneously the composition of the carrier ampholytes in the third segment was adapted to the separation demand [39,46].…”

“…The reproducibility and linearity of the pH gradient within pH range 2.0-7.5 were achieved by applying of the segmental injection [39,46]. In the first segment, the suitable spacers were dissolved in the catholyte solution; simultaneously the composition of the carrier ampholytes in the third segment was adapted to the separation demand [39,46]. Only 3% v/v solution of EtOH was used as the catholyte and the anolyte additive; the others, such as PEGs, were not used due to the possible influence on the analytical results.…”

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

“…The capillary zone electrophoretic and CIEF experiments were carried out using a laboratory-made apparatus [36] at a constant voltage of 220 kV supplied by a high-voltage unit Spellman CZE 1000 R (Plainview, NY, USA). The length of the fused-silica (FS) capillaries, 0.1 mm id and 0.25 mm od (Pliva-Lachema a. s., Brno, Czech Republic) was 350 mm, 200 mm to the detector, effective volume of the column ,1.3 mL.…”