Capillary Isoelectric Focusing and Fluorometric Detection of Proteins

Nov 3, 2006 - Pyrenebutanoate. Marie Horka´ ,*,† Filip Ru˚ zicˇ ka,‡ Jaroslav Horky´,§ Veronika Hola´ ,‡ and Karel Sˇlais†. Institute o...
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Anal. Chem. 2006, 78, 8438-8444

Capillary Isoelectric Focusing and Fluorometric Detection of Proteins and Microorganisms Dynamically Modified by Poly(ethylene glycol) Pyrenebutanoate Marie Horka´,*,† Filip Ru˚zˇicˇka,‡ Jaroslav Horky´,§ Veronika Hola´,‡ and Karel S ˇ lais†

Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverˇ´ı 97, 61142 Brno, Czech Republic, Department of Microbiology, Faculty of Medicine, Masaryk University Brno, Pekarˇska´ 53, 65691 Brno, Czech Republic, and State Phytosanitary Administration, Department of Diagnostics, Sˇ lechtitelu˚ 23, 77900 Olomouc, Czech Republic

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 oncolumn fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary. Application of electromigration techniques1 in microbiological diagnostics appears to be very useful for rapid, run-to-run reproducible separation and sensitive detection, identification, and characterization of microorganisms (MOs) in the future. The properties and behavior of MOs are more complex than those of “nonliving” analytes. The microbes, as amphoteric particles, are different in their size, shape, and composition of the outer membranes. Their isoelectric points,1-5 pI’s, are more appropriate * To whom the correspondence should be addressed. E-mail: [email protected]. Fax: (++420)-5-41212113. † Institute of Analytical Chemistry. E-mail: [email protected]. Telephone: (++420)5-3229022.; Fax: (++420)-5-41212113.. ‡ Masaryk University Brno. § State Phytosanitary Administration. (1) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465-5469. (2) Horka´, M.; Planeta, J.; Ru˚zˇicˇka, F.; Sˇlais, K. Electrophoresis 2003, 24, 13831390. (3) Kenndler, E.; Blaas, D. TrAC-Trends Anal. Chem. 2001, 20, 543-551. (4) Horka´, M.; Ru˚zˇicˇka, F.; Horky´, J.; Hola´, V.; Sˇlais, K. J. Chromatogr., B 2006, 841, 152-159.

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parameters than the electrophoretic mobilities for the characterization and identification of microbes; the pI’s are practically independent of the measurement conditions.5 Therefore, capillary isoelectric focusing (CIEF) seems to be a promising technique for the separation of MOs.1-5 The detection of microorganisms is often based on the light scattering in the UV or visible region, but it is not sensitive enough to detect low concentrations of MOs.6,7 Fluorescence detection has often been applied for the trace analysis of biological analytes. The selective adsorption of fluorophores on the cells was used in fluorescence spectroscopy,8 video fluorometry,9 fluorescence,10,11 or confocal laser microscopy,12 and in flow cytometry.12 In CIEF, the fluorogenic reagents are used for covalent or noncovalent protein labeling13 using pre-, on-, or postcolumn derivatization or “indirect” fluorometry.14 The labeling reaction must proceed rapidly, and the labeling reagent must have a low fluorescence in the unbound state and a high fluorescence enhancement when bound to biopolymers, MOs, or both. Similarly to the traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate,15 the changes of the physical-chemical properties of proteins are also expected in the fluorogenic reaction with fluorogenic reagent, e.g., 3-(2-furoyl)quinoline-2-carboxaldehyde or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.16 The ability to evaluate these changes may be essential for the efficient (5) Shen, Y.; Berger, S. J.; Smith, R. D. Anal. Chem. 2000, 72, 4603-4607. (6) Shintani, T.; Yamada, K.; Torimura, M. FEMS Microbiol. Lett. 2002, 210, 245-249. (7) Patton, W. F. BioTechniques 2000, 28, 944-957. (8) Geyer, W.; Brueggemann, L.; Flemming, I.; Nagel, B. Int. J. Syst. Bacteriol. 1991, 41, 249-254. (9) Shelly, D. C.; Warner, I. M.; Quarles, J. M. Clin. Chem. 1983, 29, 290296. (10) Yamada, K.; Torimura, M.; Kurata, S.; Kamagata, Y.; Kanagawa, T.; Kano, K.; Ikeda, T.; Yokomaku, T.; Kurane, R. Electrophoresis 2001, 22, 34133417. (11) Errampalli, D.; Leung, K.; Cassidy, M. B.; Kostrzynska, M.; Blears, M.; Lee, H.; Trevors, J. T. J. Microbiol. Methods 1999, 35, 187-199. (12) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E.; Biosens. Bioelectron. 1999, 14, 599-624. (13) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887-4894. (14) Swaile, D. F.; Sepaniak, M. J. J. Liq. Chromatogr. 1991, 14, 869-893. (15) Pinto, D. M.; Arriaga, E. A.; Craig, D.; Angelova, J.; Sharma, N.; Ahmadzadeh, H.; Dovichi, N. J.; Boulet, C. A. Anal. Chem. 1997, 69, 3015-3021. (16) Strong, R. A.; Liu, H. J.; Krull, I. S.; Cho, B. Y.; Cohen, S. A. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 1775-1807. 10.1021/ac061200h CCC: $33.50

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separation and identification of labeled proteins.17-20 For the noncovalent labeling of proteins, the fluorescent dyes, NanoOrange, Sypro red, Sypro orange, and Sypro tangerine were used. The respective isoelectric points observed do not change significantly.21,22 For the postcolumn derivatization of peptides, fluorescamine was used.23 The labeling may not only improve the detection limits but it can also change the acido-basic properties16,17 and mobilities relatively to the native species.16 And just the differences in the mobilities and isoelectric point can be used for separation, characterization, and identification of the MOs and their fragments by electrophoretic techniques.3 Several factors must be considered before the preparation of the microbial sample and the selection of the fluorogenic compounds; these include conservation of the amphoteric nature of analytes, and prevention of the aggregation of MOs and of the possibility of cell lysis,1 e.g., due to the lysis of MOs by ionic surfactants.24-27 The interactions between MOs and the additives in buffer solution,24-26,28,29 the adsorption of MOs onto the capillary wall,29-31 or both can also greatly affect the selective separation. Pyrene derivatives are known to form intermolecular excimers between two molecules in proximity.32-35 The ionic probes based on 4-(1-pyrene)butanoate (pyrenebutanoate, PB) exhibit amphiphilic behavior due to the presence of hydrophilic and hydrophobic moieties. The aggregation of PB was studied recently.32-34 Pyrenebutanoate as the anionic amphiphilic fluorescent compound was suggested as a buffer additive for dynamic modification of the proteins36 and MOs37 in capillary zone electrophoresis (CZE). In this contribution, a newly synthesized nonionogenic tenside based on pyrenebutanoate, namely, poly(ethylene glycol) 4-(1pyrene)butanoate (PB-PEG) was used in CIEF for dynamic modification and fluorometric detection of bioanalytes, proteins, (17) Richards, D. P.; Stathakis, C.; Polakowski, R.; Ahmadzadeh, H.; Dovichi, N. J. J. Chromatogr., A 1999, 853, 21-25. (18) Stoyanov, A. V.; Ahmadzadeh, H.; Krylov, S. N. J. Chromatogr., B 2002, 780, 283-287. (19) Shimura, K.; Matsumoto, H.; Kasai, K. Electrophoresis 1998, 19, 22962300. (20) Bornemann, C.; Burggraef, T.; Heimbuchel, G.; Hanisch, F. G.; Winkels, S. Anal. Bioanal. Chem. 2003, 376, 1074-1080. (21) Li, Y.; Buch, J. S.; Rosenberger, F.; De Voe, D. L.; Lee, C. S. Anal. Chem. 2004, 76, 742-748. (22) Sze, N. S. K.; Huang, T. M.; Pawliszyn, J. J. Sep. Sci. 2002, 25, 1119-1122. (23) Huff, T.; Muller, C.; Hannappel, E. Anal. Chim. Act. 1997, 352, 239-248. (24) Corradini, D. J. Chromatogr., B 1997, 699, 221-256. (25) Rabilloud, T. Electrophoresis 1996, 17, 813-829. (26) Szo ¨ko¨, E. Electrophoresis 1997, 18, 74-81. (27) Terabe, S.; Chen, N.; Otsuka, K. Micellar electrokinetic chromatography in advances in electrophoresis; VCH Publishers, Inc.: New York, 1994; Vol. 7. (28) Yao, X.-W.; Regnier, F. E. J. Chromatogr. 1993, 632, 185-193. (29) Zhao, Z.; Malik, A.; Lee, M. L. Anal. Chem. 1993, 65, 2747-2752. (30) Huang, M.; Bigelow, M.; Byers, M. LC-GC Int. 1996, 9, 658-664. (31) Ren, X.; Liu, P. Z.; Lee, M. L. J. Microcolumn Sep. 1996, 8, 529-534. (32) Nohta, H.; Satozono, H.; Koiso, K.; Yoshida, H.; Ihida, J. Yamaguchi, M. Anal. Chem. 2000, 72, 4199-4204. (33) Yoshida, H.; Nakano, Y.; Koiso, K.; Nohta, H.; Ihida, J.; Yamaguchi, M. Anal. Sci. 2001, 17, 107-112. (34) Nakano, Y.; Nohta, H.; Yoshida, H.; Saita, T.; Fujito, H.; Mori, M.; Yamaguchi, M. J. Chromatogr., B 2002, 774, 165-172. (35) Haugland, R. P. In Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Michelle, T. Z., Ed.; Molecular Probes, Inc.: Eugene, OR, 1996; p 291. (36) Horka´, M.; Sˇ lais, K. Electrophoresis 2002, 23, 1090-1095. (37) Horka´, M.; Ru˚zˇicˇka, F.; Hola´, V.; Sˇ lais, K. Electrophoresis 2005, 26, 548555.

and MOs. The fluorescent isoelectric point markers (pI markers)38 were used here for tracing of the pH gradient. The rinsing procedure between the focusing runs was shown to have a strong effect on the reproducibility4,39 and linearity of the pH gradient. Simultaneously, for safety reasons, it is necessary to disinfect the inside and the outside of the capillary after the analysis. Therefore, the segmental injection was used as in ref 4. The calculated isoelectric points of the proteins labeled with PB-PEG were compared with the pIs of the native proteins. The typical representatives of microbial strains, which cause a major part of infections in humans, 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 focused and separated using the protocol suggested here. EXPERIMENTAL SECTION Preparation of Poly(ethylene glycol) Pyrenebutanoate. The PB-PEG was prepared by the reaction from 0.38 g (1 mol L-1) of 4-(1-pyrene) butyric acid, 4 mL (10 mol L-1) of PEG (Mr 400), and 30 µL of 96% sulfuric acid. All compounds were heated for 2 h at 160 °C. The optical characteristics of the prepared nonionogenic tenside, PB-PEG, and PB were examined using PU4027 programmable fluorescence detector (Philips Scientific, Cambridge, UK). CIEF: Equipment and Procedure. The capillary isoelectric focusing experiments were carried out using the laboratory-made apparatus4041 at constant voltage (-) 20 kV supplied by highvoltage unit Spellman CZE 1000 R (Plainview, NY). During the experiments, the current decreased from 40 to 60 µA at the beginning of the experiment to 3 to 6 µA at the time of detection, depending on the sampling time interval and the sample solution. The length of the fused-silica capillaries (FS), 0.1-mm i.d. and 0.25mm o.d. (Pliva-Lachema a. s., Brno, Czech Republic), was from 230 to 270 mm, 80 mm to the detector. The ends of the fusedsilica capillary were dipped in 3-mL glass vials with the anolyte or the catholyte solutions (CaAn) and the electrodes. The PU4027 programmable fluorescence detector was modified for on-column detection. The excitation wavelength, λEX, was 335 nm, the emission wavelength, λEM, was tuned from 463 to 520 nm, and the width of the detection window was 1 mm. The light absorption (optical density) of the microbial suspensions was measured using a DU series 520 UV/visible spectrophotometer (Beckmann Instruments, Palo Alto, CA) at 550 nm. The segmental injection of the sample was accomplished by siphoning action obtained by elevating of the inletsanolyte reservoir relative to the outletscatholyte reservoir.40 The height difference of the reservoirs for sample injection, ∆h, can be adjusted in range 100-180 mm for 7-60 s. The electroosmotic flow (EOF) measurements were performed on the laboratory-made apparatus at (-) 20 kV and at constant (38) Sˇ lais, K.; Horka´, M.; Nova´cˇkova´, J.; Friedl, Z. Electrophoresis 2002, 23, 16821688. (39) Molteni, S.; Thormann, W. J. J. Chromatogr. 1993, 638, 187-193. (40) Rijnaarts, H. M. H., Norde, W., Lyklema, J., Zehnder, A. J. B. Colloids Surf. B: Biointerfaces 1995, 4, 191-197. (41) Sherbet, G.; Lakshmi, M. Biochim. Biophys. Acta 1973, 298, 50-58.

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conditions for the siphoning injection (100 mm, 5 s). The length of the whole separation fused-silica capillary (0.1-mm i.d., 0.25mm o.d.) was 320 mm, 250 mm to the on-column fluorometric detector. Association of the cells was reversed by the sonication of their suspension in Sonorex, Bandelin Electronic (Berlin, Germany). The frequency 35 kHz was used for 1 min at the temperature 30 °C. After the sonication, the microbial sample was vortexed (Vortex-Genie 2, Scientific Industries, Bohemia) 10 min and then immediately used. The detector signals were acquired and processed with the Chromatography station for Windows CSW v. 1.5, DataApex sro (Praha, Czech Republic). Chemicals. The strains included in this study, S. cerevisiae CCM 8191, E. coli CCM 3954, C. albicans CCM 8180, C. parapsilosis, C. krusei, C. glabrata, C. tropicalis CCM 8223, P. vulgaris, E. faecalis CCM 4224, S. epidermidis CCM 4418, and S. maltophilia CCM 1640 were obtained from the Czech Collection of Microorganisms. Bovine serum albumin (Mr 67 000, pI 4.938), cytochrome c, horse heart (Mr 12 400, pI 9.338), and ovalbumin (Mr 45 000, pI 4.738) were from Sigma (St. Louis, MO). Ribonuclease A from bovine pancreas (Mr 13 700, pI 8.938) was from Reanal (Budapest, Hungary) and Rhodamine B from Fluka Chemie GmbH (Buchs, Switzerland). Poly(ethylene glycol) (Mr 400 and 4000) and 4-(1-pyrene)butyric acid were from Aldrich (Milwaukee, WI). The solution of synthetic carrier ampholytes, Biolyte, pH 3-10, was obtained from Bio-Rad Laboratories (Hercules, CA). The spacers, simple ampholytes, 2-[4-(2-hydroxyethyl)-1-piperazynyl]-ethanesulfonic acid (HEPES), and L-aspartic acid (Asp) were from Merck (Darmstadt, Germany) or LOBA Chemie (Wien, Austria), respectively. All chemicals were analytical grade. Fluorescent pI markers, 5.4, 5.7, 6.0, and 6.6 were synthesized in the Institute of Analytical Chemistry.38 Other chemicals were obtained from Pliva-Lachema a.s. (Brno, Czech Republic). Safety. The potentially pathogenic MOs from risk group 2 of infectious agents were separated here at the CIEF runs. These pathogens can cause human or animal disease, but under normal circumstances, they are unlikely to be seriously hazardous to laboratory personnel. Laboratory exposures rarely cause infection leading to serious disease; effective treatment and preventive measures are available, and the risk of spread is limited. Therefore, biosafety level 2 is necessary to maintain. The inside and the outside of the capillary were disinfected by using of ethyl alcohol or other disinfecting solutions such as Persteril. All contaminated material was properly decontamined. Electrolyte Systems for CZE. For the measurement of the electroosmotic flow, 2 × 10-2 mol L-1 phosphate buffer from pH 3 to 10, PB-PEG up to 4 × 10-4 mol L-1, and Rhodamine B as a neutral marker of the electroosmotic flow were used. The excitation wavelength, λEX, was 556 nm, and the emission wavelength, λEM, was 590 nm. For all the CIEF experiments presented here, 2 × 10-2 mol -1 L sodium hydroxide and 0.1 mol L-1 orthophosphoric acid were used as the catholyte and anolyte, respectively, with addition of ethanol from zero to 3% (v/v) and PB-PEG from zero to 2 × 10-4 mol L-1. 8440

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Preparation of the Microbial Sample. The strains included in this study were inoculated on blood agar (Hi-Media, Mumbai, India) and stored at -70 °C: E. coli CCM 3954, E. faecalis CCM 4224, S. epidermidis CCM 4418, and C. albicans CCM 8180 in Itest Kryobanka B (ITEST plus, Hradec Kra´love´, Czech Republic). Before each experiment, the strains were thawed quickly at 37 °C and cultivated on Muller-Hinton’s agar (Bio-Rad, Marnes La Coquette, France) at 37 °C or on Nutrient Agar Difco (S. maltophilia CCM 1640) from Voigt Global Distribution LLC (MI) at 28 °C, both for 24 h. The microbial cultures were resuspended in physiological saline solution (PSS). The concentration of the resuspended microorganisms was estimated by the measurement of the optical density of the suspension. It was measured by spectrophotometer at 550 nm, according to the calibration curve, which was defined by reference samples. These samples were prepared by resuspending the microbial culture in PSS. The numbers of microorganisms in the reference samples were measured by dilution and by plating 100 µL of suspension on Muller-Hinton’s agar (Bio-Rad). After cultivation at 37 °C for 24 h, the colonies were counted. Composition of the Sample Pulse. The segmental injection of the sample into the capillary4 was employed here. The sample was injected in three parts. The first segment was composed of the solutions of the spacers, 2.5 × 10-4 mol L-1 HEPES, and 3.5 × 10-4 mol L-1 Asp, dissolved in the catholyte. The second segment was composed of the solution of the proteins, 10 ng mL-1, or suspension of MOs, 8 × 104-106 cell mL-1. The amount of the proteins injected into the capillary ranged from 0.5 to 1.3 pg, microorganisms from 5 to 102 cells. The fresh model mixture of proteins was dissolved in a water solution of PB-PEG with concentration (0-1) × 10-3 mol L-1. The model mixture of microorganisms additionally included 1.46 × 10-3-14.6 × 10-3 mol L-1 NaCl and 10-40% ethanol. Both sample mixtures were stored for 10-180 min at 20 °C before use. The third segment was composed of water solution of pI markers in the concentration 5 µg mL-1 (injected amounts from 1 to 15 ng), and 5% (w/v) of Biolyte. Before each injection, the capillaries were rinsed with acetone/ ethanol mixture (10:1 v/v) for 10 min and then back-flushed with the catholyte for 1 min. RESULTS AND DISCUSSION Properties of the Prepared Nonionogenic Tenside. The concentration-dependent formation of the excited-state excimers of PB-PEG is important for its practical application. According to our earlier results,36 we can expect that the optical properties of the newly synthesized nonionogenic tenside PB-PEG are similar to those of neat PB used previously.37 The emission spectra of the solutions of 1 × 10-3 mol L-1 PB, see Figure 1A, curve 1, and PB-PEG, curve 2, in phosphate buffer with 20% (v/v) EtOH and at pH 8.6 were scanned. The comparison of the maximum of fluorescence intensity of PB and PB-PEG excimers at emission wavelength, λEM, 463 nm was accomplished with the help of the PB and PB-PEG spectra normalized to λEM, 383 nm. The dependence of the ratios of the fluorescence responses of PB or PB-PEG measured at the emission wavelength 463 and 383 nm on the concentration of PB dissolved in ethanol, see curve 1, and PB-PEG dissolved in water, curve 2, respectively, are depicted in Figure 1B. According to the course of curve 2 in Figure

Figure 1. Properties of PB-PEG, the normalized spectrum (A), determination of cmc (B), and pH dependence (C). Conditions and designations, (A) the concentration of both PB (curve 1) and PBPEG (curve 2) in 2 × 10-2 mol L-1 phosphate buffer, pH 8.6, with 20% (v/v) of ethanol, cPB ) cPB-PEG, 1 × 10-3 mol L-1; λEX ) 335 nm, λEM ) 350-550 nm; (B) see (A), ethanolic solution of PB (curve 1) and water solution of PB-PEG (curve 2), cPB-PEG, cPB, 1 × 10-61 × 10-3 mol L-1; curves 1 and 2, the ratios of the fluorescence response measured at λEM 463 nm to λEM at 383 nm for PB-PEG or PB; (C) see (A), phosphate buffer; cPB-PEG, 1 × 10-4 mol L-1, λEM, 463 nm.

1B, the fluorescence intensity of PB-PEG excimers markedly increases around the range of PB-PEG concentration from 8 × 10-5 to 9 × 10-5 mol L-1. We can suppose that this concentration range corresponds with the value of the critical micellar concentration (cmc) for PB-PEG. When PB is dissolved in ethanol, the formation of PB-micelles is suppressed; see curve 1 in Figure 1B. Therefore, cmc of PB dissolved in ethanol is above 1 × 10-3 mol L-1 in comparison with cmc of PB dissolved in water, which is ∼2.5 × 10-4 mol L-1. The influence of pH on the fluorescence intensity of PB-PEG at its concentration over cmc is shown in Figure 1C. Here, the organic solvent, 20% (v/v) EtOH, is added into the samples similarly to the microbial samples procedure described below. Influence of PB-PEG on EOF. The influence of PB-PEG concentration in the electrolytes on the value of EOF is depicted in Figure 2A. The 2 × 10-2 mol L-1 phosphate buffers, pH range 3-8, without (curve 1) and with PB-PEG at the concentrations 5 × 10-5 (curve 2), 1 × 10-4 (curve 3), and 4 × 10-4 mol L-1 (curve 4) were used. Below and around micellar concentration of PBPEG in the electrolyte (curves 2 and 3), EOF decreases to 75% of the value of EOF on the bare FS capillary (curve 1) and at cPB-PEG ) 4 × 10-4 mol L-1, down to ∼50% of this value of EOF; see curve 4 versus curve 1. The decrease in EOF at the concentration of PB-PEG in the electrolyte around the cmc is sufficient for the reproducible and efficient CIEF of bioanalytes at the simulta-

Figure 2. Choice of electrolyte systems, EOF mobility (A), µEOF, and properties of pH gradient (B) as a function of pH or PB-PEG concentration, respectively. Conditions and designations, see Figure 1: (A) FS 0.1-mm i.d., 0.25-mm o.d., length 320 mm, 250 mm to the detecion cell; applied voltage (-) 20 kV; buffer, 2 × 10-2 mol L-1 phosphate buffer, cPB-PEG (mol L-1) (1) 0, (2) 5 × 10-5, (3) 1 × 10-4, and (4) 4 × 10-4; marker of EOF, Rhodamine B; ∆h, 100 mm, tinj, 5 s; λEX, 556 nm; λEM, 590 nm; (B) see (A), FS 0.1-mm i.d., length 230 mm, 80 mm to the detection cell; anolyte, 0.1 mol L-1 H3PO4; catholyte, 2 × 10-2 mol L-1 NaOH, cEtOH, CaAn, 3%(v/v); cPB-PEG, CaAn (× 10-5 mol L-1), (1, 2) 3, (3, 4) 5, (5, 6) 7, (7, 8) 10; spacers segment, 2.5 × 10-4 mol L-1 HEPES and 3.5 × 10-4 mol L-1 Asp, dissolved in catholyte, ∆h ) 180 mm; tinj, 25-s segment of pI markers; pI, 5.4, 5.7, 6.0, 6.6, and 5% (w/v) solution of Biolyte, ∆h ) 180 mm; tinj, 35 s; cPB-PEG,inj, (0-5) × 10-4 mol L-1; λEX, 335 nm, λEM, 520 nm.

neously low fluorescence intensity of the PB-PEG excimers in CaAN. Influence of PB-PEG in the Injected Sample and CaAn on the Properties of the pH Gradient. In the previous paper,4 the technique of CIEF without previous conditioning with carrier ampholytes in the pH gradient 3-10 was suggested. The problem with reproducibility and the linearity of the pH gradient was solved by applying segmental injection, where the first segment was composed of the suitable spacers, 2.5 × 10-4 mol L-1 HEPES and 3.5 × 10-4 mol L-1 Asp dissolved in 2 × 10-2 mol L-1 NaOH. This knowledge was used as the starting condition for the formation of linear pH gradient. Fluorescent pI markers 5.4, 5.7, 6.0, and 6.6 (5 µg mL-1 each) were employed to test the pH gradient. The fluorescent pI markers were detected at their maximum emission wavelength, 520 nm. 2 × 10-2 mol L-1 NaOH and 0.1 × mol L-1 H3PO4, both dissolved in 3% (v/v) EtOH, were used as the catholyte and the anolyte, respectively. The influence of the concentration of PB-PEG in CaAn, cPB-PEG, CaAn ) (0 - 1) × 10-4 mol L-1, and in the sample pulse, cPB-PEG, inj. ) (0 - 5) × 10-4 mol L-1, on the migration time of the pI marker 6.6, t6.6, and the length of the pH gradient expressed as ∆t (the difference between the migration times of the both outside pI markers, 5.46.6) was studied here; see Figure 2B. At the concentration of PBPEG in CaAn smaller than 5 × 10-5 mol L-1, see Figure 2B and Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

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Figure 3. Optimization of the incubation time, ts (min), of the cells with PB-PEG before CIEF and the dependence of the peak areas, (A) (mV s), on the number of injected cells, Ncell. Conditions and designations, see Figure 2B: cPB-PEG, CaAn, 7 × 10-5 mol L-1; sample segment - cPB-PEG,inj, 2 × 10-4 mol L-1, cEtOH,inj, 20% (v/v), 14.6 × 10-3 mol L-1 NaCl; microorganisms, C. albicans, 1 × 105 cell mL-1; ∆h, 170 mm, tinj, 14 s; λEM, 463 nm; (B) see (A), Ncell in 1 mL, 8 × 104-1 × 106; ∆h ) 100-150 mm, tinj, 7 - 14 s; ts ) 40 min.

Figure 4. Influence of the recorded emission wavelength on the high of the peaks of the microbial cells. Conditions and designations, see Figures 2B and 3: (A) sample segment - Ncells, C. albicans, C. krusei, and C. parapsilosis; in 1 mL, 8 × 104; ∆h, 170 mm, tinj, 14 s; segment of carrier ampholytes without pI markers; λEM, 500 nm; (B), see (A), segment of carrier ampholytes with pI markers, 5.4, 5.7, 6.0, 6.6; λEM, 463 nm.

curves 1 and 2, the migration time t6.6 increases and the length of the pH gradient ∆t decreases. All values are independent of the concentration of PB-PEG in the sample segment. Optimization of the Incubation Time. The cells of C. albicans, 1 × 105 in 1 mL, were incubated before CIEF, stepwise up to 180 min at 20 °C, in the solution of 2 × 10-4 mol L-1 PBPEG with 20% (v/v) of EtOH and 14.6 × 10-3 mol L-1 NaCl, see Figure 3A. For the CIEF measurements, 7 × 10-5 mol L-1 PBPEG with 3% (v/v) ethanol in the catholyte and the anolyte were used. The zones of the cells were detected at the maximum emission wavelength of the PB excimers,36 λEM ) 463 nm. The incubation time, ts, 40-60 min seems to be long enough for modification of the cells with PB-PEG in the sample before CIEF. The dependence of the peak areas of the detected zones of MOs, A, on the number of injected cells, Ncell, is in Figure 3B. The cells of Candida were incubated for 40 min before focusing runs. This dependence, A versus Ncell, is approximately linear in the range of 5-25 cells injected onto the capillary. Choice of the Suitable λEM. In this paragraph, the search for simultaneous fluorometric detection of the pI markers and PBPEG-modified microbes is discussed. The emission wavelength for the most sensitive detection of the fluorescent pI markers synthesized on the basis of fluorescein is 520 nm.38 Nevertheless, they can also be detected with lower sensitivity at λEM 480-500 nm. Thus, the range of λEM for the detection of the dynamically modified bioanalytes with PB-PEG is limited in this case. The experimental conditions for the measurements were the same as in previous experiments; see Figure 3. The sample segment was composed of 2 × 10-4 mol L-1 PB-PEG, 20%(v/v) EtOH, and 14.6 × 10-3 mol L-1 NaCl. In the catholyte and the anolyte solutions

7 × 10-5 mol L-1 PB-PEG and 3% (v/v) EtOH were dissolved. The small fluorescence response to the focused zones of the yeast strains, 8 × 104 cells mL-1, dynamically modified with PB-PEG, was detected at the emission wavelength 500 nm; see Figure 4A. Here, the fluorescent pI markers were not injected. At the maximum emission wavelength of the PB excimer, 463 nm, the zones of the modified cells, C. parapsilosis, C. krusei, and C. albicans, are detected with good fluorescence response but with zero response on the zones of the injected fluorescent pI markers 5.4-6.6 (5 µg mL-1 of each); see Figure 4B. The emission wavelength 480 nm was found as the compromise for sufficient sensitivity of detection of both the modified bioanalytes and fluorescent pI markers, see below. CIEF of pI Markers, Proteins, and Microorganisms. The electropherograms of fluorescent pI markers and dynamically modified proteins are in Figure 5A. Here, at the CIEF of the proteins, it was necessary to increase the concentrations of PBPEG in the sample segment and in CaAn to 7 × 10-4 mol L-1 and to 1 × 10-4 mol L-1, respectively, in comparison with the CIEF of microbes; see the paragraph above. The protein mixture was composed of the basic proteins, cytochrome c and ribonuclease A, and the acidic ones, albumin and ovalbumin. Their concentration was 0.01 µg mL-1 each. The detected amounts of the modified proteins were ∼1.5 pg. The values of the reported isoelectric points of the native proteins and the isoelectric points of the dynamically modified by PB-PEG are in Table 1. Significant differences between the isoelectric points of the native proteins and pI’s of the dynamically modified proteins with PB-PEG were not observed. Because of low magnitude of the observed noise, the detection limits can be expected to be at least by 2 orders of magnitude lower.

8442 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

Table 2. Isoelectric Points of MOs Reported and Dynamically Modified by PB-PEG

Figure 5. CIEF of the pI markers, proteins, and the frequent pathogens of the urinary tract infections, pH gradient 2-11. Conditions and designations, see Figure 2A, 4: (A) sample segment - cPB-PEG,inj, 7 × 10-4 mol L-1; cPB-PEG, CaAn, 1 × 10-4 mol L-1; proteins mixture, cytochrome c, ribonuclease A, albumin, and ovalbumin; ∆h of proteins, 160 mm, tinj, 14 s; λEM, 480 nm; (B) see (A) sample segment - cPB-PEG,inj, 2 × 10-4 mol L-1, cEtOH,inj, 20% (v/v); cPB-PEG, CaAn, 7 × 10-5 mol L-1; microorganism mixture, S. maltophilia, E. coli, S. epidermidis, 8 × 104 cell mL-1; λEM, 480 nm; (C) see (B) sample mixture of microorganisms, E. coli, P. vulgaris, S. epidermidis, C. albicans, and E. faecalis. Table 1. Isoelectric Points of Proteins Reported (Native) and Dynamically Modified by PB-PEG protein

reported pI38

pI of modified MOs

cytochrome c ribonuclease A albumin ovalbumin

9.3 8.9 4.9 4.7

9.25 8.83 4.85 4.62

The isoelectric points of MOs are determined by the balance between charging of anionic and cationic acid/base groups in the cell surface, together with some specific adsorption of some ions. In general, pI is an appropriate parameter complementary to cell hydrophobicity in predicting the affinity of bacteria to adhere on different surfaces.40 Nowadays, the fraction of the commonly identified microbes with known isoelectric points is negligible. In the following paragraph, we will attempt to compare our pI’s of dynamically modified microbes with known pI’s from the literature; see Table 2. The model electropherogram of CIEF of the PB-PEG modified MOs and fluorescent pI markers is in Figure 5B. The conditions for focusing are similar to those indicated earlier. The MOs were selected to focus in the wide range of the pH gradient from S. maltophilia to Gram-positive bacteria S. epidermidis. S. maltophilia, Gram-negative bacteria that sometimes colonizes on the medical implants, is a representative of strongly basic MOs, and therefore,

microorganism

reported pI

S. maltophilia E. coli S. cerevisiae

1142