Sol‐gel column technology for capillary isoelectric focusing of microorganisms and biopolymers with UV or fluorometric detection

Abstract

The sol-gel surface modification is used for capillary isoelectric focusing of microorganisms and biopolymers. The coating strongly decreases the electroosmotic flow so that it enables the use of the short capillaries down to 100 mm in the separation length. The examples of capillary isoelectric focusing of the low-molecular-mass pI markers and mixed cultures of microbial populations of Escherichia coli, Candida albicans, Staphylococcus epidermidis, and Enteroccocus faecalis with UV detection are shown. It is possible to quantify bacterial cells according to their peak areas; the minimum detectable number of microbial cells was 5 x 10(2)-1 x 10(3). The compatibility of sol-gel capillaries with sensitive fluorometric detection of fluorescent pI markers together with fluorescein labeled proteins is demonstrated.

Separation of similar yeast strains by IEF techniques

“…With regard to the supposed large numbers of gel IEF of microbial lysates, the glass etched by 1 mol/L NaOH overnight and then modified by 5% v/v g-glycidoxypropyltrimethoxysilane [46] dissolved in methanol was developed and used instead of expensive gel support films. Different from previous procedures [39,40] 0.08% Brij 35 was added to the solution of acryl-amide-Bis (5%T, 3%C), 5% w/v glycerol and simple ampholytic electrolytes, without synthetic carrier ampholytes, in the pH gradient in the pH range 2-10. The solution was degassed in the ultrasonic bath, after adding of 0.2% v/v TEMED and 0.05% w/v ammonium persulfate mixed and pipetted between the modified glass plate and the casting tray Bio-Rad Labs.…”

“…biofilm-negative strains. It may be achieved by the selection of appropriate additives to buffer solutions [26,27,[30][31][32][33] such as PEG [19,21,26,34,[36][37][38][39]. The peak shape can also be improved by the use of permanent capillary coating [39].…”

Direct and Indirect Applications of Sub- and Supercritical Water in Food-Related Analysis

“…The applications of CIEF to this purpose in this laboratory have involved the separations of a large number of microbial species and strains related to food processing and protection (Horká et al 2009a) as well as to human health (Horká et al 2003(Horká et al , 2006a(Horká et al , b, c, 2009b(Horká et al , 2011Ruzicka et al 2007), e.g., separations of plant pathogens of the Clavibacter, Xanthomonas and Pseudomonas genera (Horká et al 2009a), separation of phenotypically indistinguishable Candida species (Horká et al 2011), differentiation between biofilm-positive and biofilm-negative Staphylococcus epidermidis strains (Ruzicka et al 2007) and separations of multiple microorganisms including Candida sp. (Horká et al 2003(Horká et al , 2006a(Horká et al , b, c, 2009b, Enterococcus faecalis (Horká et al 2003(Horká et al , 2006a, Escherichia coli (Horká et al 2003(Horká et al , 2006a(Horká et al , b, c, 2009b, Klebsiela pneumoniae (Horká et al 2006b), Proteus vulgaris (Horká et al 2006b), Saccharomyces cerevisiae (Horká et al 2006c), Staphylococcus aureus (Horká et al 2006a), Staphylococcus epidermidis (Horká et al 2003(Horká et al , 2006a(Horká et al , b, c, 2009b, Streptococcus agalactiae (Horká et al 2006a, b) and Stenotrophomonas maltophilia (Horká et al 2006a, c).…”

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

“…The interactions between microorganisms (MOs) and the additives in buffer solution [1,[4][5][6][7] can also greatly affect the selective separation. The adsorption of bioanalytes can be prevented and the peak shape can be improved by the use of permanent capillary coating [8] or by the use of additives such as PEG [9][10][11][12][13][14]. PEG has also been used as the nonbonded coating for suppression of EOF in CZE [15].…”

“…Pyrene derivatives are known to form intermolecular excimer between two molecules in close proximity [26][27][28][29]. 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.…”