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 103.
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Abbreviations
- AA:
-
L-aspartic acid
- c NaOH, Ca :
-
Concentration of NaOH in the catholyte solution (mM)
- c PEG, CaAn :
-
Concentration of PEG 4000 in the catholyte and in the anolyte solutions [% (wt/vol)]
- c PEG, inj :
-
Concentration of PEG 4000 in the spacer or sample segment [% (wt/vol)]
- DSS:
-
Dilution of the stock solution of the spacer
- Glu:
-
Glutamic acid
- Δh :
-
Height difference of the reservoirs at the siphoning injection
- MAA:
-
4-morpholinyl acetic acid
- Nic:
-
Nicotinic acid
- PSS:
-
Physiological saline solution
- t :
-
Migration time (min)
- t 4.9 :
-
Migration time of the pI marker zone 4.9 (min)
- Δt :
-
Difference between the migration time of the zones of p
- TAPSO:
-
N-[tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxy-propanesulfonic acid
- t inj :
-
Injection time of the sample pulse components into the capillary (s)
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Acknowledgement
This work was supported by the Grant Agency of the Academy of Sciences of the Czech republic No. A4031302.
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Horká, M., Růžička, F., Holá, V. et al. Capillary isoelectric focusing of microorganisms in the pH range 2–5 in a dynamically modified FS capillary with UV detection. Anal Bioanal Chem 385, 840–846 (2006). https://doi.org/10.1007/s00216-006-0508-0
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DOI: https://doi.org/10.1007/s00216-006-0508-0