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New approaches to single-cell analysis by capillary electrophoresis
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New approaches to single-cell analysis by capillary electrophoresis Jennifer L. Zabzdyr, Sheri J. Lillard*
Department of Chemistry, University of California, Riverside, CA 92521-0403, USA The contents of individual cells can be readily separated by capillary electrophoresis ( CE ) and detected with a variety of methods. This article emphasizes two emerging trends of single-cell measurements by CE: ( 1 ) re¢nement in instrumentation to perform more ef¢cient analyses of individual cells and ( 2 ) detection of different classes of analytes contained in single cells. Increased interest in measuring chemistry on a cell by cell basis drives the development of instrumentation capabilities in the areas of sample handling and manipulation, automation, and detection for the improvement of fractionating and identifying intracellular contents. Detection of different classes of intracellular analytes ( compared to those studied in the earlier work ) has resulted from enhanced detector technology, improved knowledge of derivatization chemistry, and new perceptions about the measured amounts and corresponding meaning of relevant biological analytes. This article focuses on instrumentation for automated single-cell analysis and continuous cell injection. The coupling of mass spectrometry to CE for the detection and identi¢cation of analytes from individual cells is also discussed. Finally, novel methods for the detection of inositol 1,4,5-trisphosphate, RNA, and progesterone from single cells are described. z2001 Elsevier Science B.V. All rights reserved. Keywords: Capillary electrophoresis; Single-cell analysis; Intracellular analytes
1. Introduction Fields such as biomedicine stand to bene¢t considerably from the characterization of heterogene*Corresponding author. Tel.: +1 (909) 787-3392; Fax: +1 (909) 787-4713. E-mail: [email protected]
ous populations of cells. However, the low detection sensitivity of conventional biochemical methods often dictates that average, rather than distinct measurements of intracellular chemistry are performed. For example, a sample that is representative of a large population ( e.g. millions ) of cells is often subjected to an average analyte measurement, which is then extrapolated to the amount contained in one cell. Unfortunately, the assumption that this average measurement is indicative of the analyte concentration from each individual entity in the population is not always valid, especially in situations where only a few cells exhibit marked chemical differences. In these situations, a technique that meets the criteria of small sample manipulation capabilities, analyte selectivity, and detection sensitivity to measure analyte composition directly from individual cells is preferred over bulk techniques. The ¢rst example of monitoring single-cell chemistry with a separation method was published nearly 50 years ago, in which the chemistry of RNA was explored using ribonuclease digestion from individual biological cells and a dialysis-type separation [ 1 ]. This assay was then extended to separate single-cell levels of digested ribonucleotide samples using ionophoresis [ 2 ]. In 1965, a microelectrophoresis device was employed to separate hemoglobin from single, intact red blood cells [ 3 ]. Since the late 1980s, capillary electrophoresis ( CE ) in combination with various detection schemes has met the desired criteria and has emerged as a viable approach for the analysis of individual cells. Research by Kennedy et al. in 1989 demonstrated the feasibility of performing sample manipulation and separation ( CE or high performance liquid chromatography ( HPLC )) of the contents of large ( s 100 Wm diameter ) snail neurons [ 4,5 ]. This ¢rst demonstration using a microseparation technique
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to analyze large individual cells set the stage for early CE investigations that emphasized the feasibility of detecting the contents of smaller mammalian cells. Analysis of non-nucleated cells ( e.g. erythrocytes ) as samples has been described in several papers, in which different analytes have been detected with laser-induced £uorescence ( LIF ). Representative examples of measurements of red blood cells include the determination of glutathione with derivatization by monobromobimane [ 6^8 ], Na and K with indirect £uorescence [ 8,9 ] proteins with native £uorescence [ 10^12 ] or off-column derivatization [ 13 ], and enzyme-based assays of lactate dehydrogenase [ 14 ] and glutamate [ 15 ]. Although the non-nucleated feature of red blood cells affords relatively simple sample preparation and cell lysis, the majority of cells do contain a nucleus. Thus, the application of single-cell analysis by CE has expanded to include representative analyses of nucleated cells such as neurons [ 16,17 ], chromaf¢n cells [ 18,19 ] and pancreatic L-cells [ 20 ]. A look at earlier applications of CE for single-cell analysis can be found in other review articles [ 21^ 23 ]. These advances in the use of CE to analyze individual cells have led to the emergence of two signi¢cant trends in this area, which dictate the future capabilities and biological applications of this area of analytical research. The ¢rst trend is a re¢nement in instrumentation to perform more ef¢cient analyses of individual cells. The second important trend involves the ability to detect and quantitate more diverse classes of biologically important analytes [ 24^26 ].
the universality of mass spectrometry (MS ) for single-cell CE. Aided by recent improvements in sensitivity and new coupling schemes, the power of MS is manifest in its capability to monitor single-cell levels of unmodi¢ed compounds [ 33^36 ]. To illustrate these trends in instrumentational development, recent examples involving automation [ 29 ], continuous cell introduction [ 30 ], and MS detection [ 33,35,36 ] for single-cell CE are described.
2. Novel instrumentation and new detection strategies Trends in instrumentation development for single-cell analyses are seen in automation [ 27^29 ], improved sample handling capabilities [ 30 ], and improved detection strategies. For example, Li and Harrison have created a micro£uidic system on a glass chip which allows manipulation and reaction of single Saccharomyces cerevisiae, canine erythrocyte, and Escherichia coli cells within a network of channels using electroosmotic or electrophoretic pumping [ 31 ]. Zare's group has developed a sample injection method for subcellular organelles using optical trapping [ 32 ]. Many new detection strategies have taken advantage of
Fig. 1. Schematic of the injection interface for the chemical analysis of single cells by CE; ( A ) capillary holder and ( B ) vial holder. ( C ) Electropherogram of TMR-labeled species obtained from a single HT29 cell using LIF detection. Revised from [ 29 ] with permission.
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2.1. Automation Dovichi's group addressed the need for improved cell injection, lysis, and capillary conditioning by designing a multipurpose injector to automate the aforementioned parameters [ 29 ]. A schematic of their injection device is shown in Fig. 1A,B. For cell injection, this transparent device was mounted to an inverted microscope and 50 Wl of cell suspension was placed on a low attachment coverslip. The capillary holder was adjusted so that the inner bore of the capillary ( positioned vertically ) was in the center of the ¢eld of view. A cell was aligned to the inner bore by moving the microscope stage, and the capillary was lowered V10 Wm above the cell. Injection either commenced by electrokinetic or siphoning action. An electronic timer was used to control the injection and a RSD of 1% indicated that the injection time ( 1 s ) was very stable. The three-way valve that was used to control siphoning was also used to create pressure £ow in the opposite direction ( i.e. outlet to inlet ) to rinse the capillary for reconditioning of the column between electrophoretic runs. The running buffer in this experiment contained sodium dodecyl sulfate (SDS ), however, the cell suspension solution was phosphate-buffered saline (PBS ) in order to maintain cell integrity and prevent lysis. Using the carefully timed injection parameters, they found that there was a distance of only 20 Wm between the cell and the SDS interface when a 370 Wm plug of PBS ( with the cell ) was injected. Under these conditions, the cell was close enough to the detergent zone that it lysed within 30 s, without additional manipulation from the operator, i.e. injection and lysis were automatic. Electrokinetic injection was also shown to be a viable injection method, and lysis occurred similarly to siphoning-based injection. To demonstrate the utility of the optimized instrumentation, cells were incubated with a TMR ( tetramethylrhodamine )-labeled species, to measure glycosylated proteins and metabolites following separation by CE. Fig. 1C shows an electropherogram for the TMR-labeled species from a single human carcinoma cell ( HT29 ). The peak pattern ( six peaks ) correlates well with the electropherogram of a labeled cell lysate, however, intercellular variation in total £uorescence was observed ( as is typical in single-cell measurements owing to the heterogeneity of cell populations ). The detection limit of this analysis is 10321 mol of LacNAc-TMR
( c=3 ). The RSD of total £uorescence from singlecell electropherograms was 45% ( n=20 ), compared to the RSD for lysate injections of 10% ( n=18 ). Intercellular variation in total £uorescence was con¢rmed as owing to cell heterogeneity by the use of £ow cytometry, in which the RSD was found to be 49%.
2.2. Instrumentation utilizing continuous cell introduction Chen and Lillard have developed a capillary £ow injection interface that demonstrates, for the ¢rst time, continuous cell introduction and lysis, followed by migration and separation of proteins from individual human erythrocytes [ 30 ]. Optimized separation conditions show that the two major proteins in human erythrocytes, hemoglobin A0 ( Hb ) and carbonic anhydrase ( CA ), can be baseline-resolved with excellent detection limits ( 37 and 1.6 amol, respectively ). A schematic of the high-throughput set-up is shown in Fig. 2A. The device consists of two capillaries ( 21 Wm i.d.U365 Wm o.d. ) with lengths of 15 cm ( capillary A ) and 45 cm ( capillary B ). The capillaries were aligned within a 1-ml polypropylene vial using a 5-mm piece of Te£on tubing ( 1 / 16Q o.d.U0.010Q i.d. ). The inner channels of the capillaries were aligned such that a V5 Wm gap existed between them. Running buffer ( 50 mM borate, pH 9.1 ) was placed into the vial to immerse the capillary junction. The inlet end of capillary A was placed in a reservoir containing a cell suspension ( in buffer ) and the outlet of capillary B was placed in a reservoir containing buffer without cells. Cells were continuously pumped into the ¢rst capillary by electroosmotic £ow ( EOF ) following the application of +14 kV to the inlet ( the outlet was grounded ). Cell lysis was performed on-column at the junction of the two capillaries. Cellular proteins were separated by CE and detected by laser-induced native £uorescence ( with 275 nm excitation from an argon ion laser ). Because the net mobility of the cells was in the direction of EOF, this feature was used to introduce cells continuously and transport the cells to the lysis junction. Lysis was accomplished via mechanical disruption at the junction rather than by chemical lysis. As the cell was drawn out of capillary A, diffusion caused it to move in a radial direction. Because of the small inner diameter of the capillary ( 21 Wm ) and the narrow gap between the two capil-
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Fig. 2. ( A ) Schematic of instrumentation for continuous cell injection, lysis, and electrophoresis of intracellular analytes. ( B ) Electropherogram showing high-throughput CE of continuously injected individual human erythrocytes. Hb=hemoglobin A0 ; CA=carbonic anhydrase. Reprinted from [ 30 ] with permission.
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laries (V5 Wm ), the cell is siphoned into capillary B rather than diffused away. Upon entering the second capillary, the membrane is ruptured and the siphoning action sweeps the intracellular contents into the capillary. Fig. 2B shows a recursive two-peak pattern resulting from continuous introduction of human erythrocytes. Each peak pair corresponds to the major proteins from one cell. The difference between migration times of each pair ( vt = 0.62 þ 0.01 min, n = 6 ) matches well with those for a cell lysate ( vt = 0.61 þ 0.01 min, n = 3 ), con¢rming the identity of the peaks and the integrity of the system. CA and Hb in six of the 10 human erythrocytes in Fig. 2B were quantitated by external calibration ( those peaks marked with a * were excluded ). The amounts of CA per cell ranged from 4.99 to 9.78 amol, with an average 7.1 þ 2.2 amol, which is close to the literature value of 7 amol [ 37 ]. Hemoglobin levels ranged from 262 to 1025 amol per cell, with an average of 535 þ 277 amol. This is slightly higher than the reported literature value of 450 amol [ 37 ]. The average time for one cell in the high-throughput apparatus was approximately 4 min, while that for one cell in single-capillary CE was 6 min. Elimination of factors that limit throughput, such as cell sedimentation within the cell suspension, may allow more rapid analysis of individual cells.
2.3. MS detection Sweedler's group has developed an off-line detection approach for CE that utilizes matrixassisted laser desorption / ionization time-of-£ight (MALDI-TOF ) MS to detect neurochemical components ( e.g. peptides and lipids ) from individual neurons of the sea hare Aplysia californica [ 36 ]. In their experiment, a single cell is placed into a 150-nl nanovial ( constructed from a section of 0.5 mm i.d. capillary tubing ) and is lysed, then a portion of the cellular lysate is injected into the CE capillary. After elution of the intracellular components through the capillary, the eluate is deposited on a planar surface, which is used directly as the MALDI target. They demonstrated the ability to distinguish a peptide peak in the similar m / z range as lipids, by using the fractionation capabilities of CE. In contrast, when the cell was placed directly on the MALDI target without prior fractionation by CE, the lipid peaks dominated the spectrum.
Studies utilizing on-line approaches have taken advantage of the coupling aspects of the capillary outlet with electrospray ionization. The ¢rst demonstration of single-cell analysis with CE^MS was performed by a collaboration of the Smith and Ewing groups [ 33 ]. Their system sought to measure Hb in individual human erythrocytes. An individual cell was injected into an aminopropyl silane-coated capillary, lysed by osmotic pressure ( owing to the dilute running buffer ), and the hemoglobin eluted and detected under acidic conditions of 10 mM acetic acid ( pH 3.4 ). These acidic conditions are denaturing for hemoglobin, in that the peptide tetramer ( K2 L2 ) is dissociated into its constituent K and L globin chains. One peak, resulting from co-elution of the denatured K and L chains, was detected for the total ion electropherogram. The mass spectra corresponding to the peak indicated the presence of both polypeptide chains, with multiple cation adducts. These adducts revealed the high salt environment in which the cell was lysed, but nevertheless, the identity was con¢rmed. S /N s 13 was obtained for the molecular ion peaks. This study undoubtedly demonstrated the utility of CE^MS for single-cell analysis on a limited sample containing subfemtomol amounts of material. While the work by Smith and Ewing's groups utilized FTICR as the mass analyzer, a more recent approach by Cao and Moini utilized ESI^TOF for the same chemical analysis [ 35 ]. Similar electrophoresis conditions were used ( i.e. aminopropyl silane-coated capillaries with 10 mM acetic acid ( pH 3.4 ) as running buffer ). The earlier work by Smith did not have suf¢cient CE resolution to resolve the Hb chains, even though their mass spectral resolution did distinguish the two globin fractions. The experiments by Cao and Moini, however, did achieve resolution of the denatured K and L globin chains. For each single-cell run, whole blood was mixed with saline and an individual cell injected into the capillary utilizing pressure. A plug of buffer was introduced into the capillary following cell injection and allowed to incubate with the cell for V2 min to facilitate lysis. Electrophoresis revealed partial separation of the K and L globin chains, as shown in the total ion electropherogram of Fig. 3A. The broad feature of Fig. 3A was caused by the presence of the saline solution used for cell suspension, as con¢rmed by independent injections. The inset of Fig. 3A shows the reconstructed ion electropherograms for the K chain ( m / z = 842 ) and the L chain ( m / z = 883 ), revealed as peaks 1
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Fig. 3. Total ion electropherogram ( A ) and reconstructed ion electropherograms ( A, inset ) resulting from the separation of hemoglobin from an individual erythrocyte. Peak 1 of the inset refers to m / z = 842 ( K chain ) and peak 2 refers to m / z = 883 ( L chain ); +18 charge state of both ions. Mass spectra corresponding to peaks 1 and 2 are shown in ( B ) and ( C ), respectively. Reprinted from [ 35 ] with permission.
and 2, respectively. Fig. 3B,C shows the mass spectral scans of peaks 1 and 2, in which the charge distribution con¢rmed the identities of the electrophoretic peaks.
3. Novel analytes Over the years, many classes of biologically important analytes including, but not limited to ions, proteins, and amino acids, have been determined from single cells using CE coupled with a variety of detection strategies [ 6^15 ]. The expansion of single-cell investigations to include more diverse classes of analytes is revealed in recent analyses. For example, Allbritton's group has recently measured activation of £uorescently
labeled kinases in Xenopus laevis oocytes [ 38 ] and mammalian cells [ 39 ] by CE. Since a comprehensive discussion of all classes of analytes detected from single cells is beyond the scope of this paper, three representative examples were chosen: IP3 ( inositol 1,4,5-trisphosphate ) [ 24 ], RNA [ 25 ], and steroids [ 26 ].
3.1. IP3 in single Xenopus oocytes Allbritton's group has developed a novel approach for monitoring the physiologic concentration of IP3 in single cells, before and after an applied stimulus [ 24 ]. The detector set-up consisted of a cultured cell loaded with the calcium indicator dye mag-fura. The detector cell type was chosen to undergo a response ( i.e. an increase in
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Fig. 4. ( A ) Schematic of the instrumentation for rapid sampling, separation, and detection of IP3 from individual oocytes using a biological detector cell. ( B ) Measurement of endogenously produced IP3 where the ¢rst arrow indicates IP3 sampled from the oocyte cytoplasm and the second arrow indicates the calibration dose ( 5 WM ) of IP3. Reprinted from [ 24 ] with permission.
calcium release, hence an increase in £uorescence ) upon binding of IP3 to cellular receptors. Binding of IP3 to its receptor, which is located on the endoplasmic reticulum, causes release of Ca2 in the process of Ca2 signalling. An individual detector cell was isolated under the microscope and the capillary was positioned V200 Wm directly above it. ( Excitation light (V390 nm ) illuminated the cell, and £uorescence was spectrally isolated from the excitation light with an appropriate ¢lter. ) The detector operated by responding to IP3 eluting through a CE column. The detector cell was permeabilized with digitonin to facilitate entry of IP3 past the cellular membrane and into the cell. When IP3 eluted from the capillary, it bound to receptors, which caused internal calcium release and an increase in £uorescence. The separated IP3 either originated from standard solutions ( for calibration purposes ) or was sampled from oocyte cytoplasm. A calibration plot for varying concentrations of IP3 consisted of
taking the ratio of the signal for eluted IP3 divided by the signal for 5 WM IP3 ( which provided the maximal response ). This ratio method of calibration was performed for each sample concentration of IP3 and was necessary to ensure an internal calibration for each detector cell ( which show variability in their response otherwise ). For rapid sampling of oocyte cytoplasm, a piezoelectric device was used to control injection of an etched tip of the separation capillary in and out of the oocyte, as shown in Fig. 4A. A carefully controlled pulse of suction was applied when the capillary was inside the cell, and 10 nl (V1% of the cellular volume ) of cytoplasm was injected. The basal level of IP3 was determined to be 40 þ 10 nM. Fig. 4B shows the response of IP3 sampled from oocyte cytoplasm following application of lysophosphatidic acid, a physiological stimulus. The oocyte IP3 (V800 s ) and the corresponding detector cell calibration at V1350 s indicated the concentration of IP3 in this oocyte following the stimulus was 1.3 WM ( with a range of 40 nM^2 WM for different oocytes, n=8 ). The quantitative subcellular measurements using this approach provide biologically relevant detection ( i.e. detector response is related to function of the detector cell ) as well as temporal and spatial resolution (V2 s and V260 Wm, respectively ) of signal transduction chemistry in individual oocytes. By using a living cell as a biological detector, this work demonstrates an improvement in sensitivity of 10 000fold compared to other assays for IP3.
3.2. Direct sampling of RNA from single Chinese hamster ovary ( CHO ) cells Another novel analyte detectable in individual cells is RNA, which was directly sampled and separated from individual CHO ( CHO-K1 ) cells by CE in work done by Han and Lillard [ 25 ]. In these experiments, a cell was injected into the capillary, lysed on-line with SDS, and the RNA was separated ( under non-denaturing conditions ) and detected using ethidium bromide intercalation with LIF. The sieving matrix used for RNA separation contained hydroxypropylmethylcellulose and poly( vinylpyrrolidone ), to provide high separation ef¢ciency and to provide a dynamic coating on the capillary wall, respectively. Ethidium bromide was added directly to the sieving buffer for on-line intercalation into RNA. Although RNA is not a double helix like DNA, it
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Fig. 5. Electropherograms of RNA from single CHO-K1 cells ( A ) without and ( B ) with exposure to RNase in the lysing buffer. Reprinted from [ 25 ] with permission.
has the ability to fold, giving it a complex secondary structure and intramolecular base-paired regions [ 40 ]. These regions allow labeling with ethidium bromide and subsequent detection. The separation of RNA directly sampled from a single cell ( without extraction ) is shown in Fig. 5 ( trace A ). The RSD of the migration time of the last peak, over the course of six injections in two capillaries, was 0.24%. The identities of the peaks were con¢rmed as RNA in a separate experiment by introducing RNase I, an enzyme that degrades RNA into its individual bases, into the lysing buffer. Fig. 5 ( trace B ) shows the electropherogram following the introduction of RNase I, which indicates that all the peaks in trace A disappeared, hence, are identi¢ed as RNA. Based upon comparisons with literature, the ¢nal two peaks were tentatively identi¢ed as 18S and 28S rRNA, respectively [ 41 ], whereas the ¢rst group of peaks are thought to be from low molecular mass rRNA ( 5S and 5.8S fractions ) and tRNA. The amount of RNA detected roughly corresponds to 10^20 pg [ 41 ]. In this same work, Han and Lillard also investigated the use of CE^LIF to detect nucleic acid damage within single cells following exposure to hydrogen peroxide. Hydrogen peroxide is a chemical genotoxin known to cause DNA strand breaks [ 42 ]. However, it is unknown whether it or other such agents would cause damage to RNA. The cells
were incubated with different concentrations of hydrogen peroxide at room temperature for 30 min prior to injection and lysis. An area ratio was de¢ned as the peak area of the lower mass peaks divided by the peak area of the two larger mass peaks. The area ratio was used to indicate the extent of damage and is based on the hypothesis that an increased extent of nucleic acid breakage will result in a greater intensity for the lower mass fragments relative to the control studies. Based on the bellshaped dose^response curve of area ratio vs. H2 O2 concentration, the most severe damage ( greatest area ratio ) occurred following exposure to 60 mM hydrogen peroxide.
3.3. Monitoring steroids in single cultured cells Recently, Malek and Khaledi developed a method where CE with collinear LIF was used to detect and quantitate progesterone within single rat R2C cells [ 26 ]. In their system, a 20 mM SDS micellar solution was used for separation and LIF detection was accomplished using the 325 nm line of a helium^cadmium laser. Excitation and collection were accomplished using the same microscope objective, resulting in `collinear' detection. Because progesterone and other steroids do not £uoresce natively, derivatization was necessary. Malek and Khaledi chose a rather novel scheme, in which pro-
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Fig. 6. Electropherograms indicating dansylated progesterone from ( A ) an individual R2C cell and ( B ) a standard of 5U10318 mol injected. The peak eluting at V5 min is unreacted label ( dansylhydrazine ). Excitation and emission are at 325 and 515 nm, respectively. Revised from [ 26 ] with permission.
gesterone and other analyzed steroids were derivatized with dansylhydrazine, which reacts with ketone functional groups. Progesterone is a ketosteroid, that is, it has a ketone functional group, thus dansylhydrazine may be used for £uorescent tagging [ 43^45 ]. The use of a ketone-speci¢c derivatization agent prevents interference from many other cellular components, including proteins. In addition, dansylhydrazine has an excitation maximum at 340 nm, which was well-suited for the use of the 325 nm line of the helium^cadmium laser. To con¢rm that dansylhydrazine would react with common keto-steroids and that the CE^LIF apparatus could successfully detect the derivatized analytes, standard solutions of derivatized hydro-
cortisone, corticosterone, and progesterone were analyzed. In all cases, peaks corresponding to the derivatized steroid were present and clearly resolved from that of the unreacted dansylhydrazine. This CE^LIF system also provided an extensive linear range for dansylhydrazine detection. A calibration curve was constructed covering a dansylhydrazine concentration range of 5U1033 ^ 5U1039 M, with an R2 value of 0.99. The mass detection limit achieved for dansylated steroid standards using this system was 1 amol (S /N=3 ). Although the R2C cells are known to produce and secrete progesterone, Malek and Khaledi con¢rmed the steroid identity using HPLC and MS. Prior to single-cell analysis, aliquots of R2C culture media were analyzed using HPLC with UV detection. Analysis of a puri¢ed HPLC fraction con¢rmed the presence of progesterone. Derivatization of intracellular steroids was performed by incubating the R2C cells with a solution of dansylhydrazine in dimethylsulfoxide to transport the derivatizing agent into the cells. Intracellular localization of dansylhydrazine was con¢rmed by imaging the cells using £uorescence microscopy following illumination at 350 nm. Cell injection was accomplished using a micromanipulator to position the capillary inlet directly over the cell in question and using a vacuum pulse to pull the cell into the capillary. The separation buffer, which contained SDS, also acted as a lysing solution, eliminating the need for an additional lysing step. A single-cell electropherogram is shown in Fig. 6A, in which two major peaks are observed. The early peak is caused by the presence of unreacted dansylhydrazine in the cell. The second peak results from dansylated progesterone, based upon a comparison with the electropherogram of the derivatized progesterone standard ( Fig. 6B ). Based on the instrumental response to dansylated progesterone standards, the single-cell response is owing to approximately 1 amol of progesterone.
4. Future directions Since the late 1980s, fractionation and quantitation of components from individual cells have extended from demonstrations utilizing large invertebrate cells to measurements from smaller mammalian cells and even subcellular organelles. During this time the basic foundation has been established to perform these analyses with high
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sensitivity and good selectivity. In addition, a number of different intracellular analytes from a variety of cell types have been explored, owing to improvements in sample handling, detection technology, and derivatization chemistry. We believe the next generation of separations-based analyses of individual cells will encompass biological function in much greater detail than has been explored thus far, which is in part a result of enhanced biological perspectives from the analytical chemistry community. While there have been studies in the past that have tracked certain cellular functions ( e.g. stimulated release ) by CE, such studies still remain at the early stages. Each distinct cell undergoes a tremendous number of functions that rely on or elicit changes in the chemistry of the cell. The capability to monitor these subtle variations in a variety of intracellular analytes will greatly in£uence the ¢elds of biology, biochemistry, and medicine as scientists continue to unravel the chemistry of the cell and the impact this chemistry has on biology.
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[ 18 ] S.D. Gilman, A.G. Ewing, Anal. Chem. 67 ( 1995 ) 58. [ 19 ] H.T. Chang, E.S. Yeung, Anal. Chem. 67 ( 1995 ) 1079. [ 20 ] W. Tong, E.S. Yeung, J. Chromatogr. B685 ( 1996 ) 35. [ 21 ] S. Sloss, A.G. Ewing, in: J.P. Landers ( Editor ), Handb. Capillary Electrophor., CRC, Boca Raton, FL, 1994, p. 391. [ 22 ] J.A. Jankowski, S. Tracht, J.V. Sweedler, Trends Anal. Chem. 14 ( 1995 ) 170. [ 23 ] L.M. Shaner, P.R. Brown, J. Liq. Chromatogr. Relat. Technol. 23 ( 2000 ) 975. [ 24 ] V. Luzzi, C.E. Sims, J.S. Soughayer, N.L. Allbritton, J. Biol. Chem. 273 ( 1998 ) 28657. [ 25 ] F. Han, S.J. Lillard, Anal. Chem. 72 ( 2000 ) 4073. [ 26 ] A. Malek, M.G. Khaledi, Anal. Biochem. 270 ( 1999 ) 50. [ 27 ] S.N. Krylov, Z. Zhang, N.W.C. Chan, E.A. Arriaga, M.M. Palcic, N.J. Dovichi, Cytometry 37 ( 1999 ) 14. [ 28 ] Z. Zhang, S.N. Krylov, E. Arriaga, R. Polakowski, M.M. Palcic, N.J. Dovichi, Anal. Chem. 72 ( 2000 ) 318. [ 29 ] S.N. Krylov, D.A. Starke, E.A. Arriaga, Z. Zhang, N.W.C. Chan, M.M. Palcic, N.J. Dovichi, Anal. Chem. 72 ( 2000 ) 872. [ 30 ] S. Chen, S.J. Lillard, Anal. Chem. 73 ( 2001 ) 111. [ 31 ] P.C.H. Li, D.J. Harrison, Anal. Chem. 69 ( 1997 ) 1564. [ 32 ] D.T. Chiu, S.J. Lillard, R.H. Scheller, R.N. Zare, S.E. Rodr|èguez-Cruz, E.R. Williams, O. Orwar, M. Sandberg, J.A. Lundqvist, Science 279 ( 1998 ) 1190. [ 33 ] S.A. Hofstadler, J.C. Severs, R.D. Smith, F.D. Swanek, A.G. Ewing, Rapid Commun. Mass Spectrom. 10 ( 1996 ) 919. [ 34 ] G.A. Valaskovic, N.L. Kelleher, F.W. McLafferty, Science 273 ( 1996 ) 1199. [ 35 ] P. Cao, M. Moini, J. Am. Soc. Mass Spectrom. 10 ( 1999 ) 184. [ 36 ] J.S. Page, S.S. Rubakhin, J.V. Sweedler, Analyst 125 ( 2000 ) 555. [ 37 ] R.B. Pennell, in: D.M.-N. Surgenor ( Editor ), The Red Blood Cell, 2nd edn., Academic Press, New York, 1974, Chapter 3. [ 38 ] C. Lee, J. Linton, J.S. Soughayer, C.E. Sims, N.L. Allbritton, Nat. Biotechnol. 17 ( 1999 ) 759. [ 39 ] G.D. Meredith, C.E. Sims, J.S. Soughayer, N.L. Allbritton, Nat. Biotechnol. 18 ( 2000 ) 309. [ 40 ] L. Stryer, Biochemistry, 3rd edn., W.H Freeman and Company, New York, 1988, 92. [ 41 ] M. Ogura, Y. Agata, K. Watanabe, R.M. RcCormick, Y. Hamaguchi, Y. Aso, M. Mitsuhashi, Clin. Chem. 44 ( 1998 ) 2249. [ 42 ] A.R. Collins, V.L. Dobson, M. Dusinska, G. Kennedy, R. Stetina, Mutat. Res. 375 ( 1997 ) 183. [ 43 ] J.M. Anderson, Anal. Biochem. 152 ( 1986 ) 146. [ 44 ] W. Funk, R. Keller, E. Boll, V.J. Dammann, J. Chromatogr. 217 ( 1981 ) 349. [ 45 ] T. Kawasake, M. Maeda, A. Tsuji, J. Chromatogr. 232 ( 1982 ) 1.
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New approaches to single-cell analysis by capillary electrophoresis Jennifer L. Zabzdyr, Sheri J. Lillard*
Department of Chemistry, University of California, Riverside, CA 92521-0403, USA The contents of individual cells can be readily separated by capillary electrophoresis ( CE ) and detected with a variety of methods. This article emphasizes two emerging trends of single-cell measurements by CE: ( 1 ) re¢nement in instrumentation to perform more ef¢cient analyses of individual cells and ( 2 ) detection of different classes of analytes contained in single cells. Increased interest in measuring chemistry on a cell by cell basis drives the development of instrumentation capabilities in the areas of sample handling and manipulation, automation, and detection for the improvement of fractionating and identifying intracellular contents. Detection of different classes of intracellular analytes ( compared to those studied in the earlier work ) has resulted from enhanced detector technology, improved knowledge of derivatization chemistry, and new perceptions about the measured amounts and corresponding meaning of relevant biological analytes. This article focuses on instrumentation for automated single-cell analysis and continuous cell injection. The coupling of mass spectrometry to CE for the detection and identi¢cation of analytes from individual cells is also discussed. Finally, novel methods for the detection of inositol 1,4,5-trisphosphate, RNA, and progesterone from single cells are described. z2001 Elsevier Science B.V. All rights reserved. Keywords: Capillary electrophoresis; Single-cell analysis; Intracellular analytes
1. Introduction Fields such as biomedicine stand to bene¢t considerably from the characterization of heterogene*Corresponding author. Tel.: +1 (909) 787-3392; Fax: +1 (909) 787-4713. E-mail: [email protected]
ous populations of cells. However, the low detection sensitivity of conventional biochemical methods often dictates that average, rather than distinct measurements of intracellular chemistry are performed. For example, a sample that is representative of a large population ( e.g. millions ) of cells is often subjected to an average analyte measurement, which is then extrapolated to the amount contained in one cell. Unfortunately, the assumption that this average measurement is indicative of the analyte concentration from each individual entity in the population is not always valid, especially in situations where only a few cells exhibit marked chemical differences. In these situations, a technique that meets the criteria of small sample manipulation capabilities, analyte selectivity, and detection sensitivity to measure analyte composition directly from individual cells is preferred over bulk techniques. The ¢rst example of monitoring single-cell chemistry with a separation method was published nearly 50 years ago, in which the chemistry of RNA was explored using ribonuclease digestion from individual biological cells and a dialysis-type separation [ 1 ]. This assay was then extended to separate single-cell levels of digested ribonucleotide samples using ionophoresis [ 2 ]. In 1965, a microelectrophoresis device was employed to separate hemoglobin from single, intact red blood cells [ 3 ]. Since the late 1980s, capillary electrophoresis ( CE ) in combination with various detection schemes has met the desired criteria and has emerged as a viable approach for the analysis of individual cells. Research by Kennedy et al. in 1989 demonstrated the feasibility of performing sample manipulation and separation ( CE or high performance liquid chromatography ( HPLC )) of the contents of large ( s 100 Wm diameter ) snail neurons [ 4,5 ]. This ¢rst demonstration using a microseparation technique
0165-9936/01/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 0 1 ) 0 0 0 5 1 - 6
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to analyze large individual cells set the stage for early CE investigations that emphasized the feasibility of detecting the contents of smaller mammalian cells. Analysis of non-nucleated cells ( e.g. erythrocytes ) as samples has been described in several papers, in which different analytes have been detected with laser-induced £uorescence ( LIF ). Representative examples of measurements of red blood cells include the determination of glutathione with derivatization by monobromobimane [ 6^8 ], Na and K with indirect £uorescence [ 8,9 ] proteins with native £uorescence [ 10^12 ] or off-column derivatization [ 13 ], and enzyme-based assays of lactate dehydrogenase [ 14 ] and glutamate [ 15 ]. Although the non-nucleated feature of red blood cells affords relatively simple sample preparation and cell lysis, the majority of cells do contain a nucleus. Thus, the application of single-cell analysis by CE has expanded to include representative analyses of nucleated cells such as neurons [ 16,17 ], chromaf¢n cells [ 18,19 ] and pancreatic L-cells [ 20 ]. A look at earlier applications of CE for single-cell analysis can be found in other review articles [ 21^ 23 ]. These advances in the use of CE to analyze individual cells have led to the emergence of two signi¢cant trends in this area, which dictate the future capabilities and biological applications of this area of analytical research. The ¢rst trend is a re¢nement in instrumentation to perform more ef¢cient analyses of individual cells. The second important trend involves the ability to detect and quantitate more diverse classes of biologically important analytes [ 24^26 ].
the universality of mass spectrometry (MS ) for single-cell CE. Aided by recent improvements in sensitivity and new coupling schemes, the power of MS is manifest in its capability to monitor single-cell levels of unmodi¢ed compounds [ 33^36 ]. To illustrate these trends in instrumentational development, recent examples involving automation [ 29 ], continuous cell introduction [ 30 ], and MS detection [ 33,35,36 ] for single-cell CE are described.
2. Novel instrumentation and new detection strategies Trends in instrumentation development for single-cell analyses are seen in automation [ 27^29 ], improved sample handling capabilities [ 30 ], and improved detection strategies. For example, Li and Harrison have created a micro£uidic system on a glass chip which allows manipulation and reaction of single Saccharomyces cerevisiae, canine erythrocyte, and Escherichia coli cells within a network of channels using electroosmotic or electrophoretic pumping [ 31 ]. Zare's group has developed a sample injection method for subcellular organelles using optical trapping [ 32 ]. Many new detection strategies have taken advantage of
Fig. 1. Schematic of the injection interface for the chemical analysis of single cells by CE; ( A ) capillary holder and ( B ) vial holder. ( C ) Electropherogram of TMR-labeled species obtained from a single HT29 cell using LIF detection. Revised from [ 29 ] with permission.
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2.1. Automation Dovichi's group addressed the need for improved cell injection, lysis, and capillary conditioning by designing a multipurpose injector to automate the aforementioned parameters [ 29 ]. A schematic of their injection device is shown in Fig. 1A,B. For cell injection, this transparent device was mounted to an inverted microscope and 50 Wl of cell suspension was placed on a low attachment coverslip. The capillary holder was adjusted so that the inner bore of the capillary ( positioned vertically ) was in the center of the ¢eld of view. A cell was aligned to the inner bore by moving the microscope stage, and the capillary was lowered V10 Wm above the cell. Injection either commenced by electrokinetic or siphoning action. An electronic timer was used to control the injection and a RSD of 1% indicated that the injection time ( 1 s ) was very stable. The three-way valve that was used to control siphoning was also used to create pressure £ow in the opposite direction ( i.e. outlet to inlet ) to rinse the capillary for reconditioning of the column between electrophoretic runs. The running buffer in this experiment contained sodium dodecyl sulfate (SDS ), however, the cell suspension solution was phosphate-buffered saline (PBS ) in order to maintain cell integrity and prevent lysis. Using the carefully timed injection parameters, they found that there was a distance of only 20 Wm between the cell and the SDS interface when a 370 Wm plug of PBS ( with the cell ) was injected. Under these conditions, the cell was close enough to the detergent zone that it lysed within 30 s, without additional manipulation from the operator, i.e. injection and lysis were automatic. Electrokinetic injection was also shown to be a viable injection method, and lysis occurred similarly to siphoning-based injection. To demonstrate the utility of the optimized instrumentation, cells were incubated with a TMR ( tetramethylrhodamine )-labeled species, to measure glycosylated proteins and metabolites following separation by CE. Fig. 1C shows an electropherogram for the TMR-labeled species from a single human carcinoma cell ( HT29 ). The peak pattern ( six peaks ) correlates well with the electropherogram of a labeled cell lysate, however, intercellular variation in total £uorescence was observed ( as is typical in single-cell measurements owing to the heterogeneity of cell populations ). The detection limit of this analysis is 10321 mol of LacNAc-TMR
( c=3 ). The RSD of total £uorescence from singlecell electropherograms was 45% ( n=20 ), compared to the RSD for lysate injections of 10% ( n=18 ). Intercellular variation in total £uorescence was con¢rmed as owing to cell heterogeneity by the use of £ow cytometry, in which the RSD was found to be 49%.
2.2. Instrumentation utilizing continuous cell introduction Chen and Lillard have developed a capillary £ow injection interface that demonstrates, for the ¢rst time, continuous cell introduction and lysis, followed by migration and separation of proteins from individual human erythrocytes [ 30 ]. Optimized separation conditions show that the two major proteins in human erythrocytes, hemoglobin A0 ( Hb ) and carbonic anhydrase ( CA ), can be baseline-resolved with excellent detection limits ( 37 and 1.6 amol, respectively ). A schematic of the high-throughput set-up is shown in Fig. 2A. The device consists of two capillaries ( 21 Wm i.d.U365 Wm o.d. ) with lengths of 15 cm ( capillary A ) and 45 cm ( capillary B ). The capillaries were aligned within a 1-ml polypropylene vial using a 5-mm piece of Te£on tubing ( 1 / 16Q o.d.U0.010Q i.d. ). The inner channels of the capillaries were aligned such that a V5 Wm gap existed between them. Running buffer ( 50 mM borate, pH 9.1 ) was placed into the vial to immerse the capillary junction. The inlet end of capillary A was placed in a reservoir containing a cell suspension ( in buffer ) and the outlet of capillary B was placed in a reservoir containing buffer without cells. Cells were continuously pumped into the ¢rst capillary by electroosmotic £ow ( EOF ) following the application of +14 kV to the inlet ( the outlet was grounded ). Cell lysis was performed on-column at the junction of the two capillaries. Cellular proteins were separated by CE and detected by laser-induced native £uorescence ( with 275 nm excitation from an argon ion laser ). Because the net mobility of the cells was in the direction of EOF, this feature was used to introduce cells continuously and transport the cells to the lysis junction. Lysis was accomplished via mechanical disruption at the junction rather than by chemical lysis. As the cell was drawn out of capillary A, diffusion caused it to move in a radial direction. Because of the small inner diameter of the capillary ( 21 Wm ) and the narrow gap between the two capil-
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Fig. 2. ( A ) Schematic of instrumentation for continuous cell injection, lysis, and electrophoresis of intracellular analytes. ( B ) Electropherogram showing high-throughput CE of continuously injected individual human erythrocytes. Hb=hemoglobin A0 ; CA=carbonic anhydrase. Reprinted from [ 30 ] with permission.
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laries (V5 Wm ), the cell is siphoned into capillary B rather than diffused away. Upon entering the second capillary, the membrane is ruptured and the siphoning action sweeps the intracellular contents into the capillary. Fig. 2B shows a recursive two-peak pattern resulting from continuous introduction of human erythrocytes. Each peak pair corresponds to the major proteins from one cell. The difference between migration times of each pair ( vt = 0.62 þ 0.01 min, n = 6 ) matches well with those for a cell lysate ( vt = 0.61 þ 0.01 min, n = 3 ), con¢rming the identity of the peaks and the integrity of the system. CA and Hb in six of the 10 human erythrocytes in Fig. 2B were quantitated by external calibration ( those peaks marked with a * were excluded ). The amounts of CA per cell ranged from 4.99 to 9.78 amol, with an average 7.1 þ 2.2 amol, which is close to the literature value of 7 amol [ 37 ]. Hemoglobin levels ranged from 262 to 1025 amol per cell, with an average of 535 þ 277 amol. This is slightly higher than the reported literature value of 450 amol [ 37 ]. The average time for one cell in the high-throughput apparatus was approximately 4 min, while that for one cell in single-capillary CE was 6 min. Elimination of factors that limit throughput, such as cell sedimentation within the cell suspension, may allow more rapid analysis of individual cells.
2.3. MS detection Sweedler's group has developed an off-line detection approach for CE that utilizes matrixassisted laser desorption / ionization time-of-£ight (MALDI-TOF ) MS to detect neurochemical components ( e.g. peptides and lipids ) from individual neurons of the sea hare Aplysia californica [ 36 ]. In their experiment, a single cell is placed into a 150-nl nanovial ( constructed from a section of 0.5 mm i.d. capillary tubing ) and is lysed, then a portion of the cellular lysate is injected into the CE capillary. After elution of the intracellular components through the capillary, the eluate is deposited on a planar surface, which is used directly as the MALDI target. They demonstrated the ability to distinguish a peptide peak in the similar m / z range as lipids, by using the fractionation capabilities of CE. In contrast, when the cell was placed directly on the MALDI target without prior fractionation by CE, the lipid peaks dominated the spectrum.
Studies utilizing on-line approaches have taken advantage of the coupling aspects of the capillary outlet with electrospray ionization. The ¢rst demonstration of single-cell analysis with CE^MS was performed by a collaboration of the Smith and Ewing groups [ 33 ]. Their system sought to measure Hb in individual human erythrocytes. An individual cell was injected into an aminopropyl silane-coated capillary, lysed by osmotic pressure ( owing to the dilute running buffer ), and the hemoglobin eluted and detected under acidic conditions of 10 mM acetic acid ( pH 3.4 ). These acidic conditions are denaturing for hemoglobin, in that the peptide tetramer ( K2 L2 ) is dissociated into its constituent K and L globin chains. One peak, resulting from co-elution of the denatured K and L chains, was detected for the total ion electropherogram. The mass spectra corresponding to the peak indicated the presence of both polypeptide chains, with multiple cation adducts. These adducts revealed the high salt environment in which the cell was lysed, but nevertheless, the identity was con¢rmed. S /N s 13 was obtained for the molecular ion peaks. This study undoubtedly demonstrated the utility of CE^MS for single-cell analysis on a limited sample containing subfemtomol amounts of material. While the work by Smith and Ewing's groups utilized FTICR as the mass analyzer, a more recent approach by Cao and Moini utilized ESI^TOF for the same chemical analysis [ 35 ]. Similar electrophoresis conditions were used ( i.e. aminopropyl silane-coated capillaries with 10 mM acetic acid ( pH 3.4 ) as running buffer ). The earlier work by Smith did not have suf¢cient CE resolution to resolve the Hb chains, even though their mass spectral resolution did distinguish the two globin fractions. The experiments by Cao and Moini, however, did achieve resolution of the denatured K and L globin chains. For each single-cell run, whole blood was mixed with saline and an individual cell injected into the capillary utilizing pressure. A plug of buffer was introduced into the capillary following cell injection and allowed to incubate with the cell for V2 min to facilitate lysis. Electrophoresis revealed partial separation of the K and L globin chains, as shown in the total ion electropherogram of Fig. 3A. The broad feature of Fig. 3A was caused by the presence of the saline solution used for cell suspension, as con¢rmed by independent injections. The inset of Fig. 3A shows the reconstructed ion electropherograms for the K chain ( m / z = 842 ) and the L chain ( m / z = 883 ), revealed as peaks 1
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Fig. 3. Total ion electropherogram ( A ) and reconstructed ion electropherograms ( A, inset ) resulting from the separation of hemoglobin from an individual erythrocyte. Peak 1 of the inset refers to m / z = 842 ( K chain ) and peak 2 refers to m / z = 883 ( L chain ); +18 charge state of both ions. Mass spectra corresponding to peaks 1 and 2 are shown in ( B ) and ( C ), respectively. Reprinted from [ 35 ] with permission.
and 2, respectively. Fig. 3B,C shows the mass spectral scans of peaks 1 and 2, in which the charge distribution con¢rmed the identities of the electrophoretic peaks.
3. Novel analytes Over the years, many classes of biologically important analytes including, but not limited to ions, proteins, and amino acids, have been determined from single cells using CE coupled with a variety of detection strategies [ 6^15 ]. The expansion of single-cell investigations to include more diverse classes of analytes is revealed in recent analyses. For example, Allbritton's group has recently measured activation of £uorescently
labeled kinases in Xenopus laevis oocytes [ 38 ] and mammalian cells [ 39 ] by CE. Since a comprehensive discussion of all classes of analytes detected from single cells is beyond the scope of this paper, three representative examples were chosen: IP3 ( inositol 1,4,5-trisphosphate ) [ 24 ], RNA [ 25 ], and steroids [ 26 ].
3.1. IP3 in single Xenopus oocytes Allbritton's group has developed a novel approach for monitoring the physiologic concentration of IP3 in single cells, before and after an applied stimulus [ 24 ]. The detector set-up consisted of a cultured cell loaded with the calcium indicator dye mag-fura. The detector cell type was chosen to undergo a response ( i.e. an increase in
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Fig. 4. ( A ) Schematic of the instrumentation for rapid sampling, separation, and detection of IP3 from individual oocytes using a biological detector cell. ( B ) Measurement of endogenously produced IP3 where the ¢rst arrow indicates IP3 sampled from the oocyte cytoplasm and the second arrow indicates the calibration dose ( 5 WM ) of IP3. Reprinted from [ 24 ] with permission.
calcium release, hence an increase in £uorescence ) upon binding of IP3 to cellular receptors. Binding of IP3 to its receptor, which is located on the endoplasmic reticulum, causes release of Ca2 in the process of Ca2 signalling. An individual detector cell was isolated under the microscope and the capillary was positioned V200 Wm directly above it. ( Excitation light (V390 nm ) illuminated the cell, and £uorescence was spectrally isolated from the excitation light with an appropriate ¢lter. ) The detector operated by responding to IP3 eluting through a CE column. The detector cell was permeabilized with digitonin to facilitate entry of IP3 past the cellular membrane and into the cell. When IP3 eluted from the capillary, it bound to receptors, which caused internal calcium release and an increase in £uorescence. The separated IP3 either originated from standard solutions ( for calibration purposes ) or was sampled from oocyte cytoplasm. A calibration plot for varying concentrations of IP3 consisted of
taking the ratio of the signal for eluted IP3 divided by the signal for 5 WM IP3 ( which provided the maximal response ). This ratio method of calibration was performed for each sample concentration of IP3 and was necessary to ensure an internal calibration for each detector cell ( which show variability in their response otherwise ). For rapid sampling of oocyte cytoplasm, a piezoelectric device was used to control injection of an etched tip of the separation capillary in and out of the oocyte, as shown in Fig. 4A. A carefully controlled pulse of suction was applied when the capillary was inside the cell, and 10 nl (V1% of the cellular volume ) of cytoplasm was injected. The basal level of IP3 was determined to be 40 þ 10 nM. Fig. 4B shows the response of IP3 sampled from oocyte cytoplasm following application of lysophosphatidic acid, a physiological stimulus. The oocyte IP3 (V800 s ) and the corresponding detector cell calibration at V1350 s indicated the concentration of IP3 in this oocyte following the stimulus was 1.3 WM ( with a range of 40 nM^2 WM for different oocytes, n=8 ). The quantitative subcellular measurements using this approach provide biologically relevant detection ( i.e. detector response is related to function of the detector cell ) as well as temporal and spatial resolution (V2 s and V260 Wm, respectively ) of signal transduction chemistry in individual oocytes. By using a living cell as a biological detector, this work demonstrates an improvement in sensitivity of 10 000fold compared to other assays for IP3.
3.2. Direct sampling of RNA from single Chinese hamster ovary ( CHO ) cells Another novel analyte detectable in individual cells is RNA, which was directly sampled and separated from individual CHO ( CHO-K1 ) cells by CE in work done by Han and Lillard [ 25 ]. In these experiments, a cell was injected into the capillary, lysed on-line with SDS, and the RNA was separated ( under non-denaturing conditions ) and detected using ethidium bromide intercalation with LIF. The sieving matrix used for RNA separation contained hydroxypropylmethylcellulose and poly( vinylpyrrolidone ), to provide high separation ef¢ciency and to provide a dynamic coating on the capillary wall, respectively. Ethidium bromide was added directly to the sieving buffer for on-line intercalation into RNA. Although RNA is not a double helix like DNA, it
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Fig. 5. Electropherograms of RNA from single CHO-K1 cells ( A ) without and ( B ) with exposure to RNase in the lysing buffer. Reprinted from [ 25 ] with permission.
has the ability to fold, giving it a complex secondary structure and intramolecular base-paired regions [ 40 ]. These regions allow labeling with ethidium bromide and subsequent detection. The separation of RNA directly sampled from a single cell ( without extraction ) is shown in Fig. 5 ( trace A ). The RSD of the migration time of the last peak, over the course of six injections in two capillaries, was 0.24%. The identities of the peaks were con¢rmed as RNA in a separate experiment by introducing RNase I, an enzyme that degrades RNA into its individual bases, into the lysing buffer. Fig. 5 ( trace B ) shows the electropherogram following the introduction of RNase I, which indicates that all the peaks in trace A disappeared, hence, are identi¢ed as RNA. Based upon comparisons with literature, the ¢nal two peaks were tentatively identi¢ed as 18S and 28S rRNA, respectively [ 41 ], whereas the ¢rst group of peaks are thought to be from low molecular mass rRNA ( 5S and 5.8S fractions ) and tRNA. The amount of RNA detected roughly corresponds to 10^20 pg [ 41 ]. In this same work, Han and Lillard also investigated the use of CE^LIF to detect nucleic acid damage within single cells following exposure to hydrogen peroxide. Hydrogen peroxide is a chemical genotoxin known to cause DNA strand breaks [ 42 ]. However, it is unknown whether it or other such agents would cause damage to RNA. The cells
were incubated with different concentrations of hydrogen peroxide at room temperature for 30 min prior to injection and lysis. An area ratio was de¢ned as the peak area of the lower mass peaks divided by the peak area of the two larger mass peaks. The area ratio was used to indicate the extent of damage and is based on the hypothesis that an increased extent of nucleic acid breakage will result in a greater intensity for the lower mass fragments relative to the control studies. Based on the bellshaped dose^response curve of area ratio vs. H2 O2 concentration, the most severe damage ( greatest area ratio ) occurred following exposure to 60 mM hydrogen peroxide.
3.3. Monitoring steroids in single cultured cells Recently, Malek and Khaledi developed a method where CE with collinear LIF was used to detect and quantitate progesterone within single rat R2C cells [ 26 ]. In their system, a 20 mM SDS micellar solution was used for separation and LIF detection was accomplished using the 325 nm line of a helium^cadmium laser. Excitation and collection were accomplished using the same microscope objective, resulting in `collinear' detection. Because progesterone and other steroids do not £uoresce natively, derivatization was necessary. Malek and Khaledi chose a rather novel scheme, in which pro-
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Fig. 6. Electropherograms indicating dansylated progesterone from ( A ) an individual R2C cell and ( B ) a standard of 5U10318 mol injected. The peak eluting at V5 min is unreacted label ( dansylhydrazine ). Excitation and emission are at 325 and 515 nm, respectively. Revised from [ 26 ] with permission.
gesterone and other analyzed steroids were derivatized with dansylhydrazine, which reacts with ketone functional groups. Progesterone is a ketosteroid, that is, it has a ketone functional group, thus dansylhydrazine may be used for £uorescent tagging [ 43^45 ]. The use of a ketone-speci¢c derivatization agent prevents interference from many other cellular components, including proteins. In addition, dansylhydrazine has an excitation maximum at 340 nm, which was well-suited for the use of the 325 nm line of the helium^cadmium laser. To con¢rm that dansylhydrazine would react with common keto-steroids and that the CE^LIF apparatus could successfully detect the derivatized analytes, standard solutions of derivatized hydro-
cortisone, corticosterone, and progesterone were analyzed. In all cases, peaks corresponding to the derivatized steroid were present and clearly resolved from that of the unreacted dansylhydrazine. This CE^LIF system also provided an extensive linear range for dansylhydrazine detection. A calibration curve was constructed covering a dansylhydrazine concentration range of 5U1033 ^ 5U1039 M, with an R2 value of 0.99. The mass detection limit achieved for dansylated steroid standards using this system was 1 amol (S /N=3 ). Although the R2C cells are known to produce and secrete progesterone, Malek and Khaledi con¢rmed the steroid identity using HPLC and MS. Prior to single-cell analysis, aliquots of R2C culture media were analyzed using HPLC with UV detection. Analysis of a puri¢ed HPLC fraction con¢rmed the presence of progesterone. Derivatization of intracellular steroids was performed by incubating the R2C cells with a solution of dansylhydrazine in dimethylsulfoxide to transport the derivatizing agent into the cells. Intracellular localization of dansylhydrazine was con¢rmed by imaging the cells using £uorescence microscopy following illumination at 350 nm. Cell injection was accomplished using a micromanipulator to position the capillary inlet directly over the cell in question and using a vacuum pulse to pull the cell into the capillary. The separation buffer, which contained SDS, also acted as a lysing solution, eliminating the need for an additional lysing step. A single-cell electropherogram is shown in Fig. 6A, in which two major peaks are observed. The early peak is caused by the presence of unreacted dansylhydrazine in the cell. The second peak results from dansylated progesterone, based upon a comparison with the electropherogram of the derivatized progesterone standard ( Fig. 6B ). Based on the instrumental response to dansylated progesterone standards, the single-cell response is owing to approximately 1 amol of progesterone.
4. Future directions Since the late 1980s, fractionation and quantitation of components from individual cells have extended from demonstrations utilizing large invertebrate cells to measurements from smaller mammalian cells and even subcellular organelles. During this time the basic foundation has been established to perform these analyses with high
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sensitivity and good selectivity. In addition, a number of different intracellular analytes from a variety of cell types have been explored, owing to improvements in sample handling, detection technology, and derivatization chemistry. We believe the next generation of separations-based analyses of individual cells will encompass biological function in much greater detail than has been explored thus far, which is in part a result of enhanced biological perspectives from the analytical chemistry community. While there have been studies in the past that have tracked certain cellular functions ( e.g. stimulated release ) by CE, such studies still remain at the early stages. Each distinct cell undergoes a tremendous number of functions that rely on or elicit changes in the chemistry of the cell. The capability to monitor these subtle variations in a variety of intracellular analytes will greatly in£uence the ¢elds of biology, biochemistry, and medicine as scientists continue to unravel the chemistry of the cell and the impact this chemistry has on biology.
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