Anal. Chem. 2009, 81, 6897–6904
Capillary Electromigration Separation of Proteins and Microorganisms Dynamically Modified by Chromophoric Nonionogenic Surfactant Marie Horka´,*,† Filip Ru˚zˇicˇka,‡ Veronika Hola´,‡ Vladislav Kahle,† Dana Moravcova´,† and ˇ lais† Karel S Institute of Analytical Chemistry Academy of Sciences of the Czech Republic, v. v. i., Veverˇ´ı 97, 602 00 Brno, Czech Republic, and Department of Microbiology, Faculty of Medicine, Masaryk University Brno, Czech Republic A chromophoric nonionogenic surfactant poly(ethylene glycol) 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, has been prepared and used as a buffer additive for a dynamic modification of proteins and/or microorganisms including Escherichia coli, Staphylococcus epidermidis (biofilm-positive and biofilm-negative), and the strains of yeast cells Candida albicans and Candida parapsilosis (biofilm-positive and biofilm-negative) during a capillary electrophoresis and a capillary isoelectric focusing (CIEF) with UV detection at 326 nm. Values of isoelectric points of labeled proteins and microorganisms have been calculated using UV-detectable pI markers and have been found comparable with pI of the native compounds. Minimum detectable amount has been assessed lower than picograms of proteins and lower than a hundred cells injected into a separation capillary. The introduced labeling method facilitates CIEF separation of microorganisms from the clinical sample of the infected urine at their clinically important levels in the pH gradient pH range of 2-5 and their subsequent cultivation. At the same time, it has enabled the determination of albumin in human urine as a major clinical marker of urinary tract infections and kidney diseases. The fast and reliable detection and identification of etiological agents play a crucial role in selection of effective treatment of patients suffering from infectious diseases. Thus, the sensitive and effective analytical techniques are needed for the separation and detection of the low-concentrated analytes in the biological samples. These requirements are fulfilled by capillary electromigration techniques (CE) such as capillary electrophoresis (CZE) and capillary isoelectric focusing (CIEF), which are established tools utilized by clinical laboratories primarily for serum and urine protein analyses, as well as for many other clinical applications.1-3 In addition, CE, especially CIEF, appears to be suitable for a rapid * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: (++420)-5-41212113. † Institute of Analytical Chemistry Academy of Sciences of the Czech Republic. ‡ Masaryk University Brno. (1) Jenkins, M. A. Electrophoresis 2004, 25, 1555–1560. (2) Thormann, W.; Wey, A. B.; Lurie, I. S.; Gerber, H.; Byland, C.; Malik, N.; Hochmeister, M.; Gefring, C. Electrophoresis 1999, 20, 3203–3236. (3) Petersen, J. R.; Okorodudu, A. O.; Mohammad, A.; Payne, D. A. Clin. Chim. Acta 2003, 330, 1–30. 10.1021/ac900897c CCC: $40.75 2009 American Chemical Society Published on Web 07/23/2009
separation of the amphoteric bioparticles, microorganisms (MOs),4-10 and proteins11,12 conferring their pathogenicity and disease resistance.13 The conventional routinely applied microbiological methods of identification are extremely time-consuming. Electromigration separation techniques suggest a potential solution, and they usually use the differences between mobilities and isoelectric points, pI, of the separated MOs and relevant fragments like proteins for their characterization and identification.14 The concentrations of proteins and MOs in the biological/ clinical samples range from 10-11 to 10-13 mol L-1 proteins15 or tens to hundreds of cells per milliliter in blood or 103-105 cells/ mL in urine. Generally, UV absorbance as an almost universal detection system is not sensitive enough;11,16,17 thus, limits of the detection have to be improved by the online or off-line bioanalytes labeling. Both covalent and noncovalent bonding4,11,12,18-22 are recommended for the purpose of fluorometric or laser-induced fluorescence (LIF) detection.11 LIF is the most sensitive technique so far having the potential to detect proteins from 10-9 mol L-1 (4) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465–5469. (5) Kosˇt’a´l, V.; Arriaga, E. A. Electrophoresis 2008, 29, 2578–2586. (6) Desai, M. J.; Armstrong, D. W. Microbiol. Mol. Biol. Rev. 2003, 67, 38– 51. (7) Lantz, A. W.; Bao, Y.; Armstrong, D. W. Anal. Chem. 2007, 79, 1720– 1724. (8) Rodriguez, M. A.; Armstrong, D. W. J. Chromatogr., B 2004, 800, 7–25. (9) Schneiderheinze, J. M.; Armstrong, D. W.; Schulte, G.; Westenberg, D. J. FEMS Microbiol. Lett. 2000, 189, 39–44. (10) Armstrong, D. W.; Schneiderheinze, J. M.; Kullman, J. P.; He, L. FEMS Microbiol. Lett. 2001, 194, 33–37. (11) Dolnı´k, V. Electrophoresis 2006, 27, 126–141. (12) Kosˇt’a´l, V.; Katzenmeyer, J.; Arriaga, E. A. Anal. Chem. 2008, 80, 4533– 4550. (13) Mehta, A.; Brasileiro, A. C. M.; Souza, D. S. L.; Romano, E.; Campos, M. A.; Grossi-de-Sa, M. F.; Silva, M. S.; Franco, O. L.; Fragoso, R. R.; Bevitori, R.; Rocha, T. L. FEBS J. 2008, 275, 3731–3746. (14) Kenndler, E.; Blaas, D. TrAC, Trends Anal. Chem. 2001, 20, 543–551. (15) Garcı´a-Campan´a, A. M.; Taverna, M.; Fabre, H. Electrophoresis 2007, 28, 208–232. (16) Shintani, T.; Yamada, K.; Torimura, M. FEMS Microbiol. Lett. 2002, 210, 245–249. (17) Patton, W. F. BioTechniques 2000, 28, 944–957. (18) Xu, Y. H.; Li, J.; Wang, E. K. Electrophoresis 2008, 29, 1852–1858. (19) Sze, N. S. K.; Huang, T. M.; Pawliszyn, J. J. Sep. Sci. 2002, 25, 1119–1122. (20) Kremser, L.; Petsch, M.; Blaas, D.; Kenndler, E. Anal. Chem. 2004, 76, 7360–7365. (21) Welder, F.; Paul, B.; Nakazumi, H.; Yagi, S.; Colyer, C. L. J. Chromatogr., B 2003, 793, 93–105. (22) Presley, A. D.; Fuller, K. M.; Arriaga, E. A. J. Chromatogr., B 2003, 793, 141–150.
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to attomole levels15 or individual bacteria and yeast cells.7,23 The labeling may not only improve the detection limits, but it can also change their acido-basic properties24,25 and mobilities compared to the native species.24 The labeling reaction should proceed rapidly, and the respective observed isoelectric points should not change significantly.19,26 Simultaneously, it is necessary to use dynamically or statically coated capillaries for the reduction of the adsorption of the analytes to the capillary wall.9,11,15,27-30 The application of colored nonionogenic surfactants operating as dynamic modifiers may be a potential solution of these challenges. Recently, the nonionogenic surfactant based on pyrenebutanoate,31,32 namely, poly(ethylene glycol) 4-(1-pyrene)butanoate (PB-PEG) was used at CZE33 and CIEF33,34 as a buffer additive for dynamic modification and sensitive fluorimetric detection of proteins and MOs. The values of the isoelectric points of proteins and MOs labeled this way were found comparable with pI of the native compounds. In this contribution a newly prepared chromophoric (yellow) nonionogenic surfactant, poly(ethylene glycol) 3-(2-hydroxy-5-noctylphenylazo)-benzoate (HOPAB), was examined as a highly effective dynamic labeling agent for UV detection of proteins and MOs separated by CZE and CIEF. The synthesis of HOPAB was developed including determination of its basic properties. The optimized conditions for separation of the selected labeled proteins including pepsin, albumin, cytochrome c, β-lactoglobulin, ribonuclease A, and amyloglucosidase were found. Subsequently, the typical representatives of MOs causing a major part of urinary tract infectionssbacteria, Escherichia coli and Staphylococcus epidermidis biofilm-positive (+) and biofilm-negative (-), and yeast, Candida albicans and Candida parapsilosis (+) and (-)swere separated by CZE and CIEF after labeling by HOPAB. The calculated isoelectric points of proteins and MOs labeled with HOPAB were compared with pIs of native bioanalytes. Previously, bacterial pathogens were directly separated by CZE and identified from human untreated urine.35,36 In ref 35 S. saprophyticus and E. coli (the two main causative agents of urinary tract infections) were identified in urine using a direct injection technique. High efficiencies and short analysis times (
