Coupling of Solid-Phase Microextraction and Capillary Isoelectric

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Anal. Chem. 2005, 77, 165-171

Coupling of Solid-Phase Microextraction and Capillary Isoelectric Focusing with Laser-Induced Fluorescence Whole Column Imaging Detection for Protein Analysis Zhen Liu and Janusz Pawliszyn*

Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laserinduced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEFWCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, Rphycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 × 10-12 M (S/N ) 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells. Solid-phase microextraction (SPME)1-4 is a novel sampling and sample preparation technology developed in the past decade. SPME greatly simplifies chemical analysis by integrating sampling, sample preparation, and sample concentration to the solid extraction phase into a single step, with the convenient introduction of extracted analytes into an analytical instrument. SPME exhibits several apparent advantages. First, SPME is a nonexhaustive approach. The analytes can be extracted from the sample matrix before, or until, the equilibrium between the extraction phase and the sample matrix is reached; thus the extraction time is greatly reduced. Second, SPME is a solvent-free technology, a significant advantage over traditional solid-phase extraction and liquid-liquid * To whom correspondence should be addressed. E-mail: janusz@ uwaterloo.ca. Fax: +1-519-746-0435. (1) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145-8. (2) Arthur, C.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992, 64, 1187-1199. (3) Pawliszyn, J. Solid-Phase Microextraction: Theory and Practice; Wiley: New York, 1997. (4) Pawliszyn, J. Anal. Chem. 2003, 75, 2543-2558. 10.1021/ac049229d CCC: $30.25 Published on Web 11/30/2004

© 2005 American Chemical Society

extraction. Third, while the analytes are extracted, interfering molecules can be excluded. For instance, restricted access material-based SPME can extract small molecules such as drugs in blood samples but eliminate the interference of the proteins present in blood.5 Finally, this technology can be easily miniaturized to allow it to be used with both microscale analytical instruments and small living systems, such as a single cell. Recently, SPME has been demonstrated as a tool for in vivo pharmacokinetic studies.6 So far, SPME has been widely applied to environmental, pharmaceutical, clinical, forensic, and food analyses and fundamental research initiatives.7 For a single analyte extracted, SPME can be directly combined with a detection method such as spectroscopic analysis8,9 and mass spectrometry.10-12 For multiple analytes extracted, SPME can be coupled with a separation method such as gas chromatography (GC),1,2 high-performance liquid chromatography (HPLC),5,6,13 and capillary electrophoresis (CE).14-17 The most suitable separation method for coupling with SPME is GC, because of the quick desorption kinetics of analytes in gas phase at high temperature. Desorption kinetics of analytes in liquid-phase separations is generally slow, which may give rise to band broadening and analyte carryover. Capillary isoelectric focusing (CIEF) is one of the powerful CE modes, which is an equilibrium method for separating amphoteric analytes (particularly proteins) according to the differences in isoelectric points (pI).18-22 Typical resolution (5) Mullett, W. M.; Levsen, K.; Lubda, D.; Pawliszyn, J. J. Chromatogr., A 2002, 963, 325-334. (6) Lord, H.; Grant, R. P.; Walles, M.; Incledon, B.; Fahie, B.; Pawliszyn, J. B. Anal. Chem. 2003, 75, 5103-5115. (7) Pawliszyn, J., Ed. Applications of Solid-Phase Microextraction; Royal Society of Chemistry: Cambridge, U.K., 1999. (8) Merschman, S. A.; Lubbad, S. H.; Tilotta, D. C. J. Chromatogr., A 1999, 829, 377-384. (9) Wittkamp, B. L.; Hawthorne, S. B.; Tilotta, D. C. Anal. Chem. 1997, 69, 1204-1210. (10) Tong, H.; Sze, N.; Thomson, B.; Nacson S.; Pawliszyn, J. Analyst 2002, 127, 1207-1210. (11) Wang, Y.; Walles, M.; Thomson, B.; Nacson S.; Pawliszyn, J. Rapid Commun. Mass Spectrom. 2004, 18, 157-162. (12) Vas, G.; Vekey, K. J. Mass Spectrom. 2004, 39, 233-254. (13) Zambonin, C. G. Anal. Bioanal. Chem. 2003, 375, 73-80. (14) Li, S.; Weber, S. G. Anal. Chem. 1997, 69, 1217-1222. (15) Nguyen, A.-L.; Luong, J. H. Anal. Chem. 1997, 69, 1726-1731. (16) Whang, C.-W.; Pawliszyn, J. Anal. Commun. 1998, 35, 353-356. (17) Jinno, K.; Kawazoe, M.; Saito, Y.; Takeichi, T.; Hayashida, M. Electrophoresis 2001, 22, 3785-3790. (18) Hjerte´n, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270.

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Figure 1. Schematic diagram of the experimental setup for SPME-CIEF-LIF-WCID.

is 0.02 pI unit when using pH gradients formed with carrier ampholytes or 0.001 pI unit when using immobilized pH gradients. CIEF with whole column imaging detection (WCID)23-28 is a novel format of CIEF, in which amphoteric analytes are focused into narrow stationary bands. The focusing profile in CIEF-WCID at steady state does not vary significantly with focusing time. Therefore, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME. The focusing mechanism eliminates band broadening due to slow desorption, and analyte carryover can be avoided by applying an extended focusing time. In this study, we report for the first time a successful on-line coupling of SPME and CIEF with laser-induced fluorescence (LIF) WCID for protein analysis. In the SPME-CIEF-LIF-WCID method presented herein, a special cartridge is fabricated for adapting the SPME probe, and catholyte, a necessary electrolyte for CIEF, is used as the desorbing reagent. Desorption occurs simultaneously once focusing is initiated, and the desorption time can be as long as needed without any band broadening and carryover. LIF detection is chosen to lower the detection limit. Two phycobiliproteins, R- and B-phycoerythrins, were chosen as test proteins in this study for two reasons. First, these proteins are of importance in ocean sciences; they are ideal markers for investigating the distribution and trophic dynamics of picoplankton populations.29 Second, phycoerythrins have been widely used as fluorescent labels in the life sciences;30 thus, the method developed herein is applicable to phycoerythrin-labeled macromolecules, which may be of interest in other sudies. This study involved two research phases, including method development and application to real samples. In the first phase, the instrumental setup and corre(19) Liu, X.; Sosic, Z.; Krull, I. S. J. Chromatogr., A 1996, 735, 165-190. (20) Righetti, P. G.; Gelfi, C.; Conti, M. J. Chromatogr., B 1997, 699, 91-104. (21) Shen, Y.; Smith, R. D. Electrophoresis 2002, 23, 3106-3124. (22) Shimura, K. Electrophoresis 2002, 23, 3847-3857. (23) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 224-227. (24) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 2934-2941. (25) Wu, J.; Pawliszyn, J. Anal. Chem. 1994, 66, 867-873. (26) Wu, X.-Z.; Wu, J.; Pawliszyn, J. Electrophoresis 1995, 16, 1474-1978. (27) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887-4894. (28) Liu, Z.; Pawliszyn, J. J. Proteome Res. 2004, 3, 567-571. (29) Stewart, D. E.; Farmer, F. H. Limnol Oceanogr. 1984, 29, 392-397. (30) Hauglan, R. P. Handbook of Fluorescent Probes and Research Products; Molecular Probes Inc., 2002.

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sponding method were established and some basic aspects of the method were investigated. In the second phase, the usefulness of the SPME-CIEF-LIF-WCID method was demonstrated by the extraction and analysis of extracellular phycoerythrins in cultured cyanobacteria samples. EXPERIMENTAL SECTION Apparatus. The SPME-CIEF-LIF-WCID system was built inhouse on the basic design of the CIEF-LIF-WCID system described previously.27 A schematic of the instrument setup is illustrated in Figure 1. There are several differences between the old 27 and the new system used in this work: (1) the cartridge for SPME-CIEF coupling was specially designed with a SPME probe adapter within the solution junction in the catholyte reservoir for the new system; (2) a microtee was used to couple the optical fiber with the cartridge and to introduce solutions into the separation channel in the new system; (3) a ventilating fan and two ventilating holes were built onto the black box in the new system, allowing the CCD to be thermoelectrically cooled to -30 °C; and (4) the absorption detection used in the old system was removed. As in the previous system,27 the excitation wavelength was 488 nm produced by a 5-mW air-cooled argon ion laser; the baffle in the front of the laser was open only during imaging, to avoid photodecomposition of the sample. The exposure time of the CCD camera was set at 5-100 ms, depending on the fluorescence intensity of the sample. The structure of the SPME probe adapter is illustrated in Figure 2. The separation capillary was 8.4-cm-long (effective length 7.8 cm) Teflon AF 2400 tubing (167-µm i.d. and 364-µm o.d.). A piece of 3-cm-long microporous hollow fiber (380-µm i.d. and 440-µm o.d.) was connected to the separation capillary by gluing. The microporous hollow fiber was inserted into a piece of PEEK tubing (0.016-in. i.d. and 1/16-in. o.d.). The PEEK tubing was fixed 7 mm from the end of the separation capillary by gluing both ends of the PEEK tubing. Epoxy glue was applied to the surface of the hollow fiber to make a cover 6 mm long from the end of the separation capillary. Thus, there was a ∼1-mm-long uncovered microporous hollow fiber remaining, acting as a solution junction. A SPME probe can be inserted into the adapter through the hollow fiber. To prevent the separation capillary from being completely sealed by the SPME probe, an antilock function was designed by cutting the

sample injection, a common optical fiber with the same core size as the SPME probe was inserted into the adapter, and the separation capillary was filled with a sample mixture of desired concentration containing 2% Pharmarlytes (pH 3-10) and 0.5% PVP. Then, a high voltage of 1 kV was applied to the electrolyte reservoirs to begin the focusing. The whole separation column was imaged at the desired times. The SPME probe was removed from the adapter after analysis.

Figure 2. Schematic diagram of the structure of the SPME probe adapter.

Telfon AF separation capillary at an angle of 15-20°. Except for these alterations, the system was identical to the system described in previous work.27 Reagents and Materials. The cultured cyanobacteria sample CCMP833 (Synechococcus sp.) was obtained from Bigelow Laboratory for Ocean Sciences (West Boothbay Harbor, MA). RPhycoerythrin and B-phycoerythrin were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA), at a concentration of 20 mg/mL. Pharmalytes (pH 3-10, 40% in concentration), polyvinylpyrrolidone (PVP, average molecular weight about 360 000 and intrinsic viscosity 80-100 K), (3-aminopropyl)triethoxysilane (APTES) and pyrrole were obtained from Sigma (St. Louis, MO). The anolyte and catholyte were 100 mM phosphoric acid and 100 mM sodium hydroxide, respectively. Water was purified with an ultrapure water system (Barnstead/Thermolyne, Dubuque, IA). Optical fibers with 100- and 340-µm cores were purchased from Polymicro Technologies Inc (Phoenix, AZ). Celgard microporous hollow fiber with a 30-nm pore size (380-µm i.d. and 440-µm o.d.) was obtained from Hoechst Celanese (Charlotte, NC). Teflon AF 2400 capillary (167-µm i.d. and 364-µm o.d.) was purchased from Random Technologies (San Diego, CA). SPME Probe Preparation. Two types of extraction phase were used in this study, including APTES and polypyrrole (PPY). An optical fiber with a 340-µm core was first cut into suitable lengths (6 cm for the APTES phase and 7 cm for the PPY phase). One end of the fibers was then immersed into 98% sulfuric acid, heated at 120 °C for 15 min, while keeping 8 mm of the fiber below the acid surface. After the polyimide coating was removed, the fibers were washed with water for 2 min. To prepare an APTES extraction phase, the cleaned tips were immersed into a fresh solution of 5:5:90 APTES/water/ethanol for 30-90 min. Then, the prepared probes were rinsed with water for 2 min. To prepare a PPY phase, the cleaned tips were immersed in a fresh 1:1 mixture of 0.2 M ammonium persulfate solution and 0.2 M pyrrole in 1:1 water/2-propanol with stirring for 30-180 min. After the probes were rinsed with water for 2 min, they were ready for use. CIEF. The separation capillary was conditioned with water and 0.5% PVP solution for 30 min each daily. For SPME-CIEF experiments, the capillary was filled with the separation medium, 2% Pharmalytes (pH 3-10) solution containing 0.5% PVP. The SPME probe that already extracted the analytes of interest was inserted into the adapter. For CIEF experiments with conventional

RESULTS AND DISCUSSION Extraction Selectivity. Knowledge of the extraction selectivity is helpful for choosing an appropriate extraction phase for certain analytes of interest and understanding possible interferences from the sample matrix. Two extraction phases, APTES and PPY, were used in this study. The APTES phase contains primary amine groups while the PPY phase contains secondary and tertiary amine groups. Under a neutral or acidic pH condition, both extraction phases exhibit positive surface charges. Thus, both phases can be expected to act as a weak anion exchanger and be able to extract acidic proteins. As there is no long alkyl backbone or branch in these extraction phases, hydrophobic selectivity should be negligible. Therefore, weak anion exchange should be the main extraction selectivity. The test proteins, R- and B-phycoerythrins, are both acidic proteins, with literature pI values of 4.5-5.1 and 4.2-4.4, respectively.31 Under a neutral pH condition, such as in water and seawater, these proteins are negatively charged and can be extracted by both APTES and PPY extraction phases. As can be seen in the later text, both extraction phases exhibited very good selectivity for the test proteins used in this study. Cartridge Design. A simple way to couple SPME with CE is to directly insert the SPME probe into the separation capillary. The migration behaviors of desorbed analytes in such a configuration have been reported in a previous study.32 It was found that there is a sample zone-narrowing effect, the extent of which depends on the ratio of the diameter of the SPME probe to the inner diameter of the separation capillary. Such a sample zone narrowing effect is a favorable factor for SPME-CE coupling. The problems associated with this effect, however, make utilization of the effect very difficult. The zone-narrowing effect is induced by the nonuniform distribution of the electric field strength, due to the presence of the SPME probe in the separation channel. The electric field strength across the separation zone is rather low, resulting in poor separation efficiency. The high electric field in the desorption area may give rise to air bubble formation. In this study, a cartridge with a specially designed adapter was employed. The structure of the adapter has been described in the Experimental Section; the functions of the adapter are discussed here. First, the larger diameter of the adapter makes insertion of the SPME probe easier and allows for the use of a SPME probe with a larger diameter. Second, by using the adapter, the position of the SPME probe and the desorption area are confined, which is expected to provide good reproducibility. Finally, and most importantly, the extracted analytes on the SPME probe can be desorbed and transferred into the separation capillary. Before inserting the SPME probe into the adapter, the separation capillary and adapter was filled with a separation (31) http://www.martekbio.com/Fluorescent_Products/ Technical%20Bulletin%201.pdf. (32) Stoyanov, A. V.; Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 3656-3659.

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medium that can form a defined pH gradient under an electric field. After inserting the SPME probe and applying a high voltage, hydroxide ions will migrate into the adapter from the end of the extraction phase, displacing the adsorbed analytes on the SPME probe. The desorbed protein molecules are then focused to a certain position within the separation capillary where the pH equals to the pI of the protein. The cartridge designed here can be used for catholyte-assisted desorption and anolyte-assisted desorption. For anolyte-assisted desorption, reversed electrode polarity and a SPME probe with an appropriate extraction phase are needed. One important factor that should be considered when making the adapter is the size match between the adapter and SPME probe. In the previous study,27 it was noted that the volume inside the solution junction points plays a critical role in CIEF with WCID. The volume of the solution junction points must be kept as low as possible; otherwise, only a part of the pH gradient is formed within the separation capillary. Therefore, the size of the adapter and SPME probe must be chosen properly. In this study, the inner diameter of the separation channel was 167 µm, the inner diameter of the microporous hollow fiber used was 380 µm, the SPME probe had a 340 µm core size, and the thickness of the extraction phase was only several hundred nanometers (see the later text). On the basis of these data, the volume between the hollow fiber and the SPME probe within the adapter was calculated to be only 7% of the total volume between the two solution junction points. Besides, the cross-sectional area between the hollow fiber and the SPME probe within the adapter was nearly the same as that of the opening area of the separation capillary. That means that the electric field strength across the adapter was nearly the same as that across the separation capillary, avoiding nonuniform field distribution. Focusing and Desorption. There are two advantages for analyte desorption in the coupling of SPME and CIEF-WCID. One is that the desorption process is activated while the focusing process is initiated. This is different from the static desorption in SPME-HPLC,13 where desorption must be completed prior to separation. The other is that the desorption time can be as long as necessary, without considering band broadening. One characteristic of the desorption process in the SPME-CIEF-WCID method is that the volume of the desorbing reagent (hydroxide ion) can be negligible and its amount is controlled by the electric field and the separation time. The time required to ensure complete desorption of the extracted analytes was investigated. The focusing process was observed by monitoring the current through the separation channel. As shown in Figure 3, the current exponentially dropped with the separation time. The current reached a constant residual current at 15 min, which indicates the completion of the formation of the pH gradient. Usually, the focusing in CIEF is considered to be complete when the current reaches a residual current. A characteristic of completion of the focusing process is that the peak position will not change as the separation time proceeds. The dynamic focusing profiles were monitored, and the electropherograms are illustrated in Figure 4. It is clear that the desorbed analyte molecules are gradually focused to a position where the pH equals to the pI of the protein, while the peak position did not vary after 15 min. The total separation time was first set at 20 min, and then another run was 168 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

Figure 3. Dependence of the current through the separation channel on separation time. Extraction phase, 460-nm-thick APTES; sample, 2 µg/mL R-phycoerythrin in water; extraction, 30 min with stirring; separation medium, 2% Pharmalytes (pH 3-10) containing 0.5% PVP; applied voltage, 1 kV.

Figure 4. Dynamic focusing process in the SPME-CIEF. Conditions were identical to those described in Figure 3.

carried out with the same SPME probe without further extraction, to determine whether a 20-min separation time was long enough to ensure that no carryover resulted. The electropherograms obtained from the two separations are shown in Figure 5A. It is apparent that a small portion of the extracted protein molecules still remained on the SPME probe after the first separation, resulting in a small peak in the second separation. By comparing the peak area of the two runs, the protein amount in the second run was estimated to be 3% of the total amount of the extracted protein. Thus, the originally set separation time was not long enough and must be extended to ensure complete desorption. The separation time was extended to 30 min, and the electropherograms for two consecutive runs for a single extraction with the same SPME probe are shown in Figure 5B. There was no detectable peak in the second run, indicating complete desorption within 30 min. The effect of the extraction-phase thickness on the desorption time was investigated with APTES-phase SPME probes of different thickness. The extraction phases were prepared with two different reaction times, 30 and 90 min. As expected, the thickness measured by the scanning electron microscope increased with

Figure 6. Dependence of the peak area on the extraction time. Extraction phase, 200-nm-thick APTES; sample, 2 µg/mL R-phycoerythrin in water; extraction, 30 min with stirring; separation medium, 2% Pharmalytes (pH 3-10) containing 0.5% PVP; applied voltage, 1 kV.

Figure 5. CIEF profiles at 20 (A) and 30 min (B) for two consecutive runs of a single extraction with the same SPME probe. Conditions were identical to those described in Figure 3.

the reaction time, 200 nm for 30-min reaction time and 460 nm for 90-min reaction times. When the thinner extraction phase was used, the time required for complete desorption was found to be 20 min. In comparison, when the thicker extraction phase was used, the time required for complete desorption was longer (30 min). Thus, it is clear that a thin extraction phase is favorable for fast desorption. Extraction Equilibrium. The extraction equilibrium was investigated by plotting the peak area for the extracted protein against the extraction time. As shown in Figure 6, an equilibrium was reached in 10 min, under the conditions used. Therefore, for the sample investigated and with the SPME probe used, only 10 min was needed for equilibrium extraction. The extraction can be lower than 10 min for preequilibrium extractions. Detection Sensitivity. Figure 7 illustrates the electropherograms for extracted R- and B-phycoerythrin from 0.4 nM Rphycoerythrin and 8 nM B-phycoerythrin in water. The detection sensitivity was excellent for R-phycoerythrin, offering a limit of detection (LOD) of 3.5 × 10-12 M (S/N ) 3). Such a detection sensitivity is much better than the result reported in CE-LIF analysis with direct sample injection (4.2 × 10-10 M).33 The (33) Viskari, P. J.; Kinkade, C. S.; Colyer, C. L. Electrophoresis 2001, 22, 23272335.

Figure 7. Electopherograms for SPME-CIEF-LIF-WCID of Rphycoerythrin (A) and B-phycoerythrin (B). Extraction phase, PPY; sample, (A) 0.1 µg/mL R-phycoerythrin in water, (B) 2 µg/mL B-phycoerythrin in water; extraction, (A) 15 min with stirring, (B) 35 min with stirring; separation medium, 2% Pharmalytes (pH 3-10) containing 0.5% PVP; applied voltage, 1 kV.

detection sensitivity for B-phycoerythrin was not as good, giving a LOD of 8.3 × 10-10 M (S/N ) 3). The reason is because the excitation wavelength (488 nm) was not optimal for the detection of B-phycoerythrin. B-Phycoerythrin has a maximum adsorption at 565 nm and a secondary maximum adsorption 546 nm. In comparison, R-phycoerythrin has a maximum adsorption at 565 nm but a secondary maximum adsorption at 480 nm. Reproducibility. The reproducibility of the method was investigated with APTES-phase SPME probes and 8 nM Rphycoerythrin in water. The run-to-run reproducibility was good, with a relative standard deviation (RSD) value of 2% (n ) 6) for peak position and 13% (n ) 6) for peak area. The reproducibility was comparable to that of CIEF-LIF-WCID with sample injection. The probe-to-probe reproducibility for peak position was also good, giving a RSD value of 2% (n ) 6). However, the probe-to-probe reproducibility for peak area was poor, with a RSD value of 47% (n ) 4). The probe-to-probe reproducibility can be further improved through controlling crucial factors in the probe preparaAnalytical Chemistry, Vol. 77, No. 1, January 1, 2005

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Figure 8. CIEF-LIF-WCID of the CCMP833 cyanobacteria with conventional sample injection. Sample mixture, 100-fold diluted CCMP833 containing 2% Pharmalytes (pH 3-10) and 0.5% PVP; applied voltage, 1kV.

Figure 10. CIEF profiles for SPME extracted extracellular phycoerythrins from the cultured cyanobacteria sample (A) and from the R-phycoerythrin spiked cultured cyanobacteria sample (B). Extraction phase, PPY; sample, (A) CCMP833, (B) CCMP833 spiked with 2 µg/ mL R-phycoerythrin; extraction, 10 min with stirring; separation medium, 2% Pharmalytes (pH 3-10) containing 0.5% PVP; applied voltage, 1 kV.

tion procedure such as coating thickness, surface area, surface roughness, and so on. Analysis of Real Sample. Cyanobacteria, which produce phycobiliproteins, are recognized as an important and widespread component of marine picophytoplankton that contribute significantly to total carbon biomass and primary productivity of the oceans.34,35 Some methods have been developed to extract29,36 and determine33,37 intracellular phycobiliproteins in cyanobacteria. In this study, our interest was to extract and analyze extracellular phycoerythrins in cyanobacteria samples, because extracellular proteins can reveal the viability of cells. The change in membrane permeability forms the basis for traditional cell viability assays that use colorimetric or fluorescent dyes.38,39 When a cell is alive, its cell membrane is intact, so only small ions or molecules can

permeate the cell wall. When a cell is dead, the cell membrane becomes permeable to large molecules, and thus intracellular proteins will leak out of the cell. In a recent paper,40 extracelluar proteins were suggested as signaling markers for the assessment of cell viability. Analysis of extracellular proteins can be easily performed with CE separation with standard sample injection. However, sampling and transportation of bacteria samples may be problematic. If the samples contain living cells, cell death during the storage and transportation processes will make the analysis results unreliable. An alternative method is to remove the cells by filtering; however, such a procedure may result in protein loss. In such a case, the SPME-CIEF method should be a good analysis tool. Extracellular proteins can be extracted onsite with SPME, and then the SPME probes are sealed in a SPME assembly and transported to the laboratory for analysis. To determine the efficacy of using the SPME-CIEF method for the analysis of extracellular proteins, the cultured cyanobacteria sample was first examined with conventional sample injection. Figure 8 shows the electropherogram for a cyanobacteria sample diluted 100-fold with the separation medium. Two overlapped peaks were observed: one for the extracellular phycoerythrins and the other for the cyanobacteria cells. The peak identities were confirmed by comparison with CIEF of homogenized cyanobacteria under the otherwise identical conditions, which gave a dramatically increased peak for the extracellular proteins and a significantly reduced peak for the bacteria (data not shown). The high starting current in the CIEF experiment for injected samples (3 times higher than that of the SPME-CIEF experiment) indicated the presence of a large amount of various inorganic salts in the culture medium. SPME-CIEF-LIF-WCID analysis was then carried out with SPME probes of APTES and PPY phases. Figure 9 shows

(34) Waterbury, J. B.; Watson, S. W.; Guillard, R. R. L.; Brand, L. E. Nature 1979, 277, 293-294. (35) Johnson, P. W.; Sieburth, J. M. Limnol. Oceanogr. 1979, 24, 928-935. (36) Viskari, P. J.; Colyer, C. L. Anal. Biochem. 2003, 319, 263-271. (37) Wyman, M. Limnol. Oceanogr. 1992, 37, 1300-1306.

(38) Darzynkiewicz, Z.; Li, X.; Gong, J. P. Assays of cell viability: discrimination of cells dying by apotosis, Methods in Cell Biology; Academic Press: New York, 1994. (39) King, M. A. J. Immunol. Methods 2000, 243, 155-166. (40) Liu, Z.; Pawliszyn, J. Anal. Biochem.. in press.

Figure 9. CIEF profiles for SPME extracted extracellular phycoerythrins from the cultured cyanobacteria sample (A) and a blank (B). Extraction phase, 200-nm-thick APTES; sample, (A) CCMP833, (B) none; extraction, (A) 15, (B) 0 min; separation medium, 2% Pharmalytes (pH 3-10) containing 0.5% PVP; applied voltage, 1 kV.

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the CIEF profile of extracted extracellular phycoerythrins by APTES-phase SPME. Clearly, only the extracellular phycoerythrins were extracted while the cyanobacteria cells remained in the sample matrix. The extracellular phycoerythrins in the cyanobacteria sample were quantitatively estimated by the standard addition method with a PPY-phase SPME probe. As shown in Figure 10, the R-phycoerythrin spiked sample exhibited an increased peak area. By comparing the peak areas, the extracellular phycoerythrins in the cyanobacteria sample tested were estimated to correspond to 2 nM R-phycoerythrin. Thus, the usefulness of the SPME-CIEF-LIF-WCID method for the analysis of the extracellular phycoerythrins in cyanobacteria samples was demonstrated. It should be pointed out that R- and B-phycoerythrins were not differentiated with the CIEF-LIF-WCID method presented, as they are isomers with identical pI values. However, this can be resolved by using fluorescence spectra scanning. CONCLUSIONS The on-line coupling of SPME and CIEF has been implemented for the first time by coupling with LIF-WCID. The developed approach has been successfully applied to the extraction and analysis of trace extracellular phycoerythrins in cultured cyanobacteria samples. CIEF-WCID is the most compatible liquid-phase analytical method for coupling with SPME. By using the specially designed cartridge, the extracted analytes on the SPME probe are continuously desorbed and focused into individual bands under the electric field in CIEF-WCID. The desorption time can be as (41) Wu, J.; Li, S.-C.; Watson, A. J. Chromatogr.. A 1998, 817, 163-171. (42) Malinshi, K.; Taha, Z. Nature 1992, 358, 676-678. (43) Wightman, R. M.; Finnegan, J. M.; Pihel, K. Trends Anal. Chem. 1995, 14, 154-158. (44) Huang, L.; Kennedy, R. T. Trends Anal. Chem. 1995, 14, 158-164. (45) Troyer, K. P.; Wightman, R. M. Anal. Chem. 2002, 74, 5370-5375. (46) Hoch H. C., Jelinshi, L., Craighead, H. G., Eds. Nanofabrication and Biosystems: intergrating materials science, engineering, and biology; Cambridge University Press: New York, 1996. (47) Huang, W.-H.; Zhang, L.-Y.; Cheng, W.; Pang, D.-W.; Wang, Z.-L.; Cheng, J.-K. Gaodeng Xuexiao Huaxue Xuebao 2003, 24, 425-427.

long as necessary, without any band broadening and analyte carryover. This method proved to be simple, quick, highly efficient, and highly sensitive for protein analysis. The naturally fluorescent proteins phycoerythrins at nanomolar level in real samples were extracted and analyzed within 30 min, avoiding interference from the sample matrix. The total analysis time can be shortened by reducing the sizes of the separation channel and the SPME probe and by using a higher voltage. In CIEF with UV adsorption WCID on a commercial instrument, with a 100-µm-i.d. separation channel and an applied voltage of 3 kV, typical analysis time ranges from 2 to 5 min.41 Thus, it is possible to reduce the total analysis time to within 5 min. If the size of the separation channel and the SPME probe can be further miniaturized to the nanometer scale, when nanoscale SPME is coupled with nanochip-based CIEF-LIF-WCID, the total analysis time can be expected to be only a few seconds. On the other hand, by combining with an on-fiber or on-line fluorescent derivatization approach, applications of the SPMECIEF-LIF-WCID method can be expanded for nonfluorescent proteins. Along with the rapid development of materials science and engineering, it can be expected that a nanoscale SPME-CIEFLIF-WCID system can be applied to single-cell proteomic analysis in the future. As there have been successful applications of microelectrode- and nanoelectrode-based voltammetry in singlecell analyses,42-47 such an approach should be feasible. ACKNOWLEDGMENT The authors acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC). L.Z. acknowledges Dr. Christa Colyer at the University of Technology Institute of Ontario for the valuable discussions.

Received for review May 26, 2004. Accepted October 13, 2004. AC049229D

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