Capillary Isoelectric Focusing and Fluorometric Detection of Proteins and Microorganisms Dynamically Modified by Poly(ethylene glycol) Pyrenebutanoate

Abstract

The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.

Recent advances in the analysis of biological particles by capillary electrophoresis

“…The same research group developed a fluorescence detection scheme based on the labeling of 4-[(1-pyrene)butanoate] (PB), which forms a fluorescent intermolecular excimer with amino groups on the surface of the microorganisms being analyzed, rendering them fluorescent after their electrophoretic separation [27]. In an exciting development, the same group later combined the PB detection scheme with CIEF [28]. Here, they synthesized PEG-PB and this molecule played dual role as both a dynamic coating and a fluorescent reagent, making it possible to detect down to ten cells.…”

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

“…MAA was prepared by the reaction of morpholin and chloracetic acid (Sigma) and PB-PEG by the reaction of 4-(1-pyrene) butyric acid and PEG 400 [32]. The specifications [39,40] of the used spacers, simple ampholytes, are described in ref.…”

“…[32], the proteins modified by PB-PEG were adsorbed on the capillary surface at CZE separation. Therefore, the dynamic modification of the inner surface of the capillary by PEG was necessary.…”

“…The composition of CaAn in CIEF in the pH gradient range of 2-5.5 was different from that introduced in ref. [32], the separation of the dynamically modified proteins and MOs by the nonionogenic tenside PB-PEG at CIEF in the pH gradient range of 3-10 with fluorometric detection. Similar to CZE of proteins the addition of PEG was necessary as prevention of the protein adsorption.…”

“…Since the pI's [8,9,13,30,31] of MOs are practically independent of the measurement conditions [31] and seem to be very effective for their identification, CIEF could be the predominant electromigration technique for the separation of cells in future. The nonionogenic tenside based on pyrenebutanoate, namely PEG 4-(1-pyrene)butanoate (PB-PEG), was used in CIEF for dynamic modification and fluorimetric detection of bioanalytes, proteins, and MOs [32] in the pH gradient in the range of 3-10. The rinsing procedure between the focusing runs was shown to have a strong effect on the reproducibility [13,33] and linearity of the pH gradient.…”