Anal. Chem. 1995,67,58-64
Analysis of Single Cells by Capillary Electrophoresis with On=ColumnDerivatization and Lasermhduced Fluorescence Detection S. Douglas8 Oilman and Andrew 0. Ewing* 152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802
On-columnderivatization of single mammalian cells with capillary electrophoretic separation and laser-induced fluorescence detection is described. Individual cells are electrophorectically injected into the front end of the separation capillary, which is used as a chamber to lyse the cell and derivatize its contents for subsequent separation and detection. Reagents to lyse the cell and derivatize its contents are electrophoreticallyinjected into the front end of the capillary (2.7 mm,600-pL volume for a 17pm4.d. capillary) after the cell has been injected. Dopamine and five amino acids have been quantitatively determined in individual rat pheochromocytoma cells after on-column derivatization with naphthalene-2,3-&carboxaldehyde and CN-. Average values of compounds determined in these cells range from 180 f 110 amoV cell for aspartic acid to 5.1 f 1.5 fmoVcell for taurine. Capillary electrophoresis (CE) has emerged in recent years as a versatile tool for the analysis of chemical species ranging from small metallic cations to proteins and DNA,-4 The ability of CE to sample extremely small volumes and to selectively determine multiple analytes using the technique's high efficiency and resolving power has led to the application of CE to the analysis of the chemical contents of single cell^.^-^^ While the sampling and separation capabilities of CE are well-suited to the analysis of the wide variety of molecules found in cells, species are typically (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981,53,1298-1302. (2) Manning, C. A; Kennedy, R T. Anal. Chem. 1994,66,280R-314R (3) Weinberger, R Practical Capillary Electrophoresis; Academic: San Diego, CA, 1993. (4) Capillary Electrophoresis Technology; Guzman, N. A, Ed.; Marcel Dekker: New York, 1993. (5) Wallingford, R A; Ewing, A G. Anal. Chem. 1988,60,1972-1975. (6) Kennedy, R T.; Oates, M. D.; Copper, B. R; Nickerson, B.; Jorgenson, J. W. Science 1989,246,57-63. (7) Chien, J. B.; Wallingford, R A; Ewing, A G. J. Neurochem. 1990,54,633638. (8) Olefirowicz, T.M.; Ewing, A G. Anal. Chem. 1990,62, 1872-1876. (9) Olefirowicz, T. M.; Ewing, A G. J. Neuvosci. Methods 1990,34,11-15. (10) Olefirowicz, T. M.; Ewing, A G. Chimia 1991,45,106-108. (11) Ewing, A G.; Strein, T. G.; Lau,Y. Y. Acc. Chem. Res. 1992,25, 440-447. (12) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992,64,2841-2845. (13) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992,64,3045-3051. (14) Ewing, A G. J. Neurosci. Methods 1993,48,215-224. (15) Hoyt, A M., Jr.; Beale, S. C.; h a n n , J. P., Jr.; Jorgenson, J. W. 1. Microcolumn Sep. 1993,5,325-330. (16) Xue, Q.; Yeung, E. S. J. Chromatogr. A 1994,661,287-295. (17) Ktistensen, H. IC;Lau, Y. Y.; Ewing, A G. J. Neurosci. Methods 1994,51, 183-188. (18) Xue, Q.; Yeung, E. S. Anal. Chem. 1994,66,1175-1178. (19) Rosenzweig, Z.; Yeung, E. S. Anal. Chem. 1994,66,1771-1776. (20) Bergquist, J.; Tarkowski, A; Ekman, R ; Ewing, A Proc. Natl. Acad. Sci. U.SA., in press.
58 Analytical Chemistry, Vol. 67,No. 1, January 7, 7995
present in single cells at femtomole to zeptomole levels (10-15mol), making quantitative detection of these compounds extremely challenging. Electrochemical laserinduced fluorescence &IF) detection,12,13,15,16,18 and immunoa~say~~ have all been used to obtain sufficientlylow mass detection limits for quantitative determination of compounds in individual cells by CE. The simplest detection schemes used for single-cell analysis by CE take advantage of native properties of analytes. Electrochemical detection has been used with CE to detect electroactive neurotransmitters in single whole snail neurons,1°J7in cytoplasmic injections from snail neuron^,^,^-^^ and in single whole human lymphocytes.2O Hemoglobin and carbonic anhydrase in individual human erythrocytes have been separated by CE and quantitated using native fluorescence detection induced with a W line from an Ar ion 1 a ~ e r . lThe ~ activities of several lactate dehydrogenase isoenzymes have been determined in individual human erythrocytes by monitoring the enzyme-catalyzed production of NADH using LIF detection after separation of the isoenzymes by CE.18 Unfortunately,many compounds found in single cells are not easily detected in their native state, and methods must be developed to enhance detection of these analytes. Covalent labeling with a fluorophore is an attractive method for enhancing detection sensitivity for nonfluorescent analytes due to the low detection limits that can be obtained using LIF detection with CE,21-24the large number of derivatizing reagents that are a ~ a i l a b l e ,and ~ ~ the , ~ ~wide variety of analytes that can be derivatized using these reagent^.^^^^^ Despite the availability of fluorescent labels, difficulties in sample handling limit the use of derivatization for single-cell analysis. It is desirable to minimize dilution of the contents of a single cell during derivatization in order to maintain favorable kinetics for the labeling reaction and to avoid diluting analytes that are already present at trace levels. However, the volumes of cells analyzed by CE range from L for large invertebrate neurons5to 10-13 L for erythrocytes.12 The addition of reagents for derivatization without serious dilution of the contents of a single cell is extremely challenging with the current technology for manipulating solutions. (21) Sweedler, J. V.; Shear, J. B.; Fishman, H. A; Zare, R N.; Scheller, R H. Anal. Chem. 1991,63,496-502. (22) Hernandez, L.; Escalona, J.; Joshi, N.; Guzman, N. J. Chromatogr. 1991, 559,183-196. (23) Wu, S.;Dovichi, N. J. Tulantu 1992,39,173-178. (24) Lee, T. T.; Yeung, E. S. J. Chromatogr. 1992,595,319-325. (25) Detection-Oriented Derivatization Techniques in Liquid Chromatography; Lingeman, H., Underberg, W. J. M., Eds.; Marcel Dekker: New York, 1990. (26) Szulc, M. E.; Krull, I. S. J. Chromutogr. A 1994,659,231-245. 0003-2700/95/0367-0058$9.00/0 0 1994 American Chemical Society
Two approacheshave been used successfullyfor derivatization of the contents of single cells for subsequent analysis by CE with LIF detection. Thiols in single erythrocytes have been derivatized by incubating living cells with a derivatizing reagent, monobromobimane, to which the cell membrane is permeable.12 The cell itself acts as a subpicoliter reaction chamber, and attomole levels of glutathione have been determined in erythrocytes using this method with CE and LIF detection.lZ Alternatively, single cells have been physically isolated and placed in 20@nLmicrovials for d e r i ~ a t i z a t i o n . ~Using J ~ ~ ~ ~these microvials and micropipet manipulation of nanoliter volume solutions,B individual snail neurons6 and bovine adrenal medullary ce1lsl5have been derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), while total volumes of derivatized cell contents have been limited to 20-30 nL. A large number of peaks have been observed for both cell types using CE with LIF detection.6'15 This same sampling and derivatization methodolgy has been k e d with open tubular liquid chromatography and electrochemical detection to determine amino acids quantitatively in individual snail neurons at femtomole levels.27 This paper describes a method for on-column derivatization of single mammalian cells for analysis by CE with LIF detection. The front end of the separation capillary is used as a derivatization chamber, where the cell and lysing/derivatizing buffer are electrophoreticallyintroduced and mixed. After completion of the reaction in the front of the capillary, derivatized analytes are separated by CE. This method for single-cell derivatization reduces the requiments for micromanipulation to injection of the cell only, while the capillary confines analytes and reagents to a small volume, minimizing dilution. Derivatized amines from individual rat pheochromocytoma (PC12) cells have been separated by CE with LIF detection, and a number of analytes in these cells have been quantitated at femtomole and attomole levels.
has been described previ0usly.2~Briefly, positive high voltage was applied at the injection end of the capillary through a platinum wire in the sample buffer reservoir using a 30-kV dc power supply (Bertan Associates, Inc., Hicksville, NY). The detection end of the capillary was grounded through a platinum wire in the detection buffer reservoir. The high-voltage electrode and sample buffer reservoir were enclosed in a Plexiglas interlock box for operator safety. Experiments were carried out using a 17-pmi.d./360-pm-o.d. fused-silica capillary (Polymicro Technologies; Phoenix, AZ). Capillary lengths were 100 cm, and the detection window was located 85 cm from the injection end of the capillary. All separations were performed in 100 mM borate buffer at pH 9.5, using a 3@kV separation potential. The electrophoretic current was typically 3.3 pA. To aid cell injection, the injection ends of the capillaries were etched with hydrofluoric acid using a procedure similar to that described for construction of microinjectors for cytoplasmic injection! The polyimide coating (3 mm) was removed from the end of the capillary, and the exposed end was immersed in HF (5095 aqueous solution) while He flowed through the capillary (100 psi). Capillaries were etched until the outer diameter was 30-50 pm (-90 min). The etched end of the capillary was briefly immersed in a saturated NaZC03 solution
to neutralize HF and was then rinsed in doubly distilled water. The etched end was cleaved under a microscope with a scalpel at a point where the inner diameter was 25-40 mm, yielding a conical structure. Capillaries were filled with separation buffer using a stainless steel reservoir with applied He pressure (50100 psi). Buffer solutions were filtered with a 0.2-pm filter (Alltech, Deerfield, IL) prior to use. Detection Apparatus. The LIF detection system was identical to that described previously3O except that the optics were adjusted for use with the 442-nm beam (50 mw) from a He-Cd laser (Model 4240NB, Idconix, Santa Clara, CA). The beam was attenuated to 1.5 mW with a neutral density filter (Model 50282, Oriel Corp., Stratford, CT) and was focused onto the capillary using a 12.7-mm-focal-length, fused-silica, plan0 convex lens (Oriel). Fluorescence was collected with a 20x microscope objective lens (Oriel). The image of the collected fluorescence was focused on a 2.5" spatial filter and was passed through an interference filter centered at 490 nm (490DF30; Omega Optical, Brattleboro, VT) before reaching the photomultiplier tube (Rl527, Hamamatsu Corp., Bridgewater, NJ), which was biased at -1000 V. The photocurrentwas monitored using a Keithley Instruments 427 current amplifier (Cleveland, OH) with the rise time set at 300 ms. The output from the current amplifier was sent to a System Gold Analog Interface Module 406 (Beckman Instruments, Fullerton, CA) operating at a data conversion rate of 5 Hz. Data were collected and analyzed with an IBM PS/2 (Model 502) using System Gold software (Beckman). Chemicals. All chemicals were used as received from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Naphthalene-2,3-dicarboxaldehyde, NaCN, and digitonin were obtained from Fluka Chemical Corp. (Ronkonkoma, NY). DL-Glycine, taurine, and HF (50% aqueous solution) were obtained from Aldrich Chemical Co. (Milwaukee, WI), and (L)-(+)-glutamic acid was obtained from Fisher Scientific Co. (Fair Lawn, NJ). All buffers were prepared in doubly distilled water (Coming MegaPure MP-3A purification system, Coming, NY). Borate buffer solutions were made by adjusting the pH of boric acid solutions to the desired pH by addition of NaOH. A stock solution of NaCN (50 mM> was prepared in 50 mM pH 9.5 borate buffer, and a stock solution of digitonin (1.0 mM) was prepared in ethanol. Stock solutions of NDA (50 mM) were prepared weekly in acetonitrile, and these solutions were stored in the dark. Stock solutions of y-Glu-Gly (1.0 mM) and Val-Tyr-Val (3.0 mM) were prepared weekly in doubly distilled water. Amino acid stock solutions were prepared in borate buffer (100 mM, pH 9.5). Catecholamine stock solutions were prepared as 1.0 mM solutions in 100 mM perchloric acid. All stock solutions except NaCN were refrigerated when not in use. PC12 Cell Cultures. Seed PC12 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained as described by Greene and T i ~ c h l e r .Cells ~ ~ were incubated at 37 "C, 8% COZin 6@ x 15mm, 5 pg/cm2 mouse collagen IV coated (Collaborative Biomedical Products, Bedford, MA) Falcon culture dishes (Becton Dickinson Labware) until they reached near confluence. (L)-Ascorbic acid (0.015 g/L) was added into the culture medium after each cell propagation. Growth medium was composed of 84%phenol red-free RPMI-1640 (Sigma)
(27) Oates, M. D.; Cooper, B. R; Jorgenson, J. W. Anal. Chem. 1990,62,15731577. (28) Kennedy, R T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524. (29) Wallingford, R A; Ewing, A G. Anal. Chem. 1987, 59, 1762-1766.
(30) Gilman, S. D.; Pietron, J. J.; Ewing, A G. J Microcolumn Sep. 1994, 6, 373-384. (31) Greene, L. A; Tischler, A S . Proc. Nafl.Acad. Sci. U S A . 1976, 73, 24242428.
EXPERIMENTAL SECTION CE Apparatus. The CE system used was built in-house and
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
59
supplemented with 10%heat-inactivated horse serum, 5% fetal bovine serum URH Bioscience, Lenexa, KS), and 1%antibiotic antimycotic solution (Sigma). The cell medium was changed every 2-3 days. Cells were studied between passages 1and 2 in the undifferentiated condition. Precolumn Derivahtion. Precolumn derivatization of standard solutions for constructing calibration curves, determinining peak identities, and determining reaction times was conducted in glass vials (2.0 mL). A final volume of 1.9 mL was used for each experiment. Appropriate volumes of analyte stock solutions were diluted in separation buffer, NaCN was added ( h a l concentration 1.0 mM), and then NDA was added (final concentration, 1.0 mM). Samples were allowed to react for at least 10 min prior to injection. Samples contained 2% acetonitrile due to the addition of NDA from a stock solution in acetonitrile. Determination of the reaction time for analytes was performed using the same derivatization procedure. Concentrations used were as follow: Val-Tyr-Val, 5.0 x M; norepinephrine (NE), 2.0 x M; dopamine @A), 1.0 x M; Gly, 3.75 x M; y-Glu-Gly,6.2 x M; Asp, 1.9 x M. Samples were injected (5 s at 30 kv) at a minimum of three different times (typically 5.0, 7.0, and 10.0 min) after addition of NDA, and peak areas at each reaction time (n = 3 for each reaction time) were compared to determine approximately when each reaction was completed. On-Column Derivatization of Single Cells. The medium from a culture dish of PC12 cells was removed by aspiration, rinsed three times with warm (room temperature) Ca2+-free balanced salt solution, and replaced with 5 mL of the Ca2+-free solution. This solution (PH7.4) consisted of the following: NaC1, 150 mM; KCl, 4.2 mM; MgC12,2.7 mM; NaH2P04,l mM; glucose, 11.2 mM; HEPES 4(2-hydroxyethyl)-piperazineethanesulfonic acid), 10 mM. A medium that was Ca2+-freewas used in order to eliminate release of compounds in the cell by Ca2+dependent e x o c y t ~ s i s . ~y-Glu-Gly ~ - ~ ~ was used as an internal standard to account for cell medium injected with the cell and was added to the dish of cultured cells to give a concentration of 1.0 x M. The injection end of the capillary was attached to a micromanipulator (E. Leitz, Inc., Rockleigh, NJ) and positioned at the bottom of the culture dish. The detection end of the capillary was kept level with the bottom of the culture dish to eliminate gravity flow during the experiment. An SV8 stereomicroscope (Carl Zeiss, Inc., Thornwood, NY) was used for cell injections, and the culture dish was illuminated from underneath using an Intralux 6000 illuminator (Volpi Manufacturing USA, Aubrun, NY) . A single PC12 cell was observed at approximately 200x magnitication, and the capillary was placed near the cell. A potential of 2 kV was applied to the culture dish via a platinum wire, and the tip of the capillary was used to gently detach the cell from the culture dish. The capillary tip was then positioned so that the cell was drawn into the capillary by electroosmotic flow as observed under the microscope. The total injection time ranged from 15 to 90 s. The capillary was then carefully moved from the culture dish into a reservoir (1 mL) containing a buffer to derivatize and lyse the cell, and a 3@s, 2.0-kV injection of this buffer was made. Fresh reagent buffer was prepared immediately (32) Pozzan, T.; Gatti, G.; Dozio, N.; Wcentini, L. M.; Meldolesi, J. ]. Cell Bioi. 1984, 99, 628-638. (33) Leszczyszyn, D. J.; Jankowski,J. A; Viveros, 0. H.; Diliberto, E. J., Jr.; Near, J. A; Wightman, R M.]. Eiol. Chem. 1990,265, 14736-14737. (34) Chen, T. IC: Luo, G.: Ewing, A G. Anal. Chem. 1994, 66, 3031-3035. (35) Chen, T. IC: Ewing, A. G. submitted for publication in]. Neurosci. Methods.
60 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
before each experiment and consisted of digitonin (50 pM),NDA (2.5 mM), NaCN (2.5 mM), and Val-Tyr-Val(3.0 x M, internal standard) in the separation buffer. The buffer also contained 5% ethanol and 5%acetonitrile from the stock solutions of digitonin and NDA, respectively. The cell was allowed to lyse and react for 10.0 min, and a second injection (15 s at 2.0 kv) of the reagent buffer was made to ensure that no sample was lost during transfer of the capillary to the separation buffer reservoir in the interlock box. The capillary was then returned to a reservoir containing the separation buffer, and a separation voltage of 30 kV was applied. Peak identities were determined by examining electrophoretic mobilities and by spiking the reagent used for on-column derivatization with standards. Analyses to determine background amine levels in the cell culture medium were performed before and after every two PC12 cell injections. The injection end of the capillary was manipulated to the bottom of the cell culture dish to an area where no cells were present. A 30-s injection was then made at 2.0 kV, and the lysing/derivatizing solution was injected and allowed to react with the injected cell medium exactly as described for cell injections. Separation and detection were as described for the cell injection experiments. The reagent buffer used for on-column derivatization experiments was injected separately to determine if this reagent produced any peaks in the cell electropherograms. Only one peak was observed, which was attributed to the reaction of the derivatization reagent with an amine impurity, and this peak was determined to be neutral (10.0-10.6 min in cell electropherograms). Calibration Curves and Data Treatment. Peak areas were used for all quantitativemeasurements and were determined using System Gold software. Analytes in cell injections and cell background injection experiments were quantitated on the basis of linear calibration curves spanning the range of concentrations found in cell experiments and consisting of points of at least four different concentrations for each analyte (n = 3 at each concentration). The curves were determined by derivatizing analytes at each concentration used to construct the curve, and standard leastsquares analysis gave linear correlation coefficients greater than 0.998 for all analytes examined except for Glu (0.991). The internal standard y-Glu-Gly was used to determine the quantities of analytes injected in the cell medium along with the cell, and Val-Tyr-Val was used to standardize detector sensitivity and the derivatization reaction. The amounts of analytes determined to have been injected in the medium with the cell on the basis of the background determinations were substracted from the quantities of the analytes determined in the cell experiments. Calculated injected amounts for anions and cations were corrected for bias due to electrokinetic injection except in cell experiments. Separation efficiencies were calculated on the basis of measurement of the peak width at 10%of the peak height. All errors are reported as f the standard error of the mean. Mety Considerations. Electrophoretic injection of cells from a culture dish involves the application of high voltage (2.0 kv) outside of the protective Plexiglas interlock box, and due caution should be used to avoid exposure to high voltage. Extreme care must be taken when working with HF as it can cause severe bums. Hydrofluoroic acid should be neutralized with Na~C03prior to disposal. Caution should also be used when working with solutions containing cyanide.
RESULTS AND DISCUSSION On-Column Derivatization of Single Cells. A key aspect
of this work was the procedure for injection and oncolumn derivatization of single cells. The first part of the method, dislodging the cell from the bottom of the culture dish with the capillary tip and drawing it inside the capillary using electroosmotic flow, is the only step in the procedure that requires the use of a microscope and micromanipulation. Detachment of the cell from the culture dish and injection of the cell into the capillary is carefully monitored under the microscope at approximately 200x magnification. It is important to visually observe the cell moving into the bore of the capillary, as cells that appear to have disappeared into the capillary tip are often only adhering to the underside of the capillary, where they are obscured from view. In the second step of the procedure, the capillary containing the cell is carefully removed from the culture dish and placed in a reservoir containing reagents to lyse the cell and derivatize its contents. The reagents are electrophoretically injected into the capillary. The adhesion of the cell to the capillary wall is advantageous in this case, as it impedes migration of the cell further into the capillary so that injected reagent is pulled over and past the injected cell. The third part of this procedure is the most critical part of the injection and derivatization method. The reagents are allowed time to lyse the cell and derivatize the contents of the cell for LIF detection after CE separation. The injection end of the capillary acts as a reaction chamber for the cell, limiting dilution of the contents of the lysed cell and dilution of the reagents that derivatize the cell's contents (vide infra). Analysis of Single PC12 Cells. The PC12 clonal cell line has been shown to be a versatile tool in n e u r o ~ c i e n c e .The ~~~~~ cell line has been used extensively as a model system to study neuronal differentiation, the actions of nerve growth factor, and catecholamine biosynthesis and secretion.36 The PC12 cell line is being used in our research as a model system for the study of vesicular release of dopamine from single cells using ampercmetric detection with microelectrode^.^^^^ The development of methodology for studying the chemical composition of the cells used in these studies is therefore of interest. Electropherograms of the contents of individual PC12 cells obtained using on-column derivatization with NDNCN- and LIF detection are shown in Figures 1 and 2. Several of the peaks in the elctropherograms from single PC12 cells have been identified on the basis of their electrophoreticmobilities and by spiking the reagent buffer used for cell derivatization with standard solutions of analytes. The migration times of the peaks in the electropherograms increase after several cell injections as has been observed previously with analysis of single cells by CE.9 The quantities of five compounds determined in six individual PC12 cells are shown in Table 1.The electropherogram in Figure lA is from cell 3 in Table 1, and the electropherogram in Figure 1B is from a control injection of the cell medium performed immediately after cell 3 and displayed at the same scale. It is clear that many of the compounds present in the cell electropherogramare also present in the electropherogram of the cell medium, and these peaks will interfere with quantitative determination of the contents of these cells. In order to quantitate compounds in single PC12 cells, the contribution to the single-cell electropherograms of species present in cell medium must be considered. An internal standard, (36) Guroff, G. In Cell Culture in the Neurosciences; Bottenstein, J. E.,Sato, G., Eds.; Plenum: New York, 1985 pp 245-272.
10
16
13
19
22
25
2a
22
2s
28
Time (min)
I
10
16
13
19
Time (min)
Figure 1. (A) Electropherogram of the NDA-derivatized contents from a single PC12 cell (cell 3). Val-Tyr-Val and y-Glu-Gly are internal standards. Unidentified peaks are labeled U, and taurine is T. (B) Electropherogram of a control injection of the medium from the cell culture dish from which the PC12 cell in A was injected. The vertical scale is identical to that in A.
10
11
12
13
14
Time (min)
15
16
17
Figure 2. Expanded view of an electropherogram from a single PC12 cell (cell 4). The dopamine peak is labeled DA. The nearly vertical line immediately following peak 7 is the front part of the large, narrow, unidentified peak found in the single-PClP-cell electropherograms (peak 3 in Figure 1A). Peaks were identified by comparison of electrophoretic mobilitiesto a standard electropherogram. The rate of electroosmotic flow differed from that in Figure 1.
y-Glu-Gly,is added to the balanced salt solution bathing the cell, and this internal standard is used to determine the amount of cell medium injected with the PC12 cells. Taurine, glycine, and glutamic acid levels in the electropherogramsof cell medium are typically found at less than 30%of the levels observed in singlecell electropherograms. Alanine and aspartic acid are sometimes found at similar levels in the cell and background experiments. Dopamine, which can be observed in an expanded view of a singlecell electropherogram (Figure 2), and norepinephrine have not been detected in any of the background experiments. The relative amounts of analytes found in the cell medium do not reflect the results obtained for cell separations. For example, the ratio of Analytical Chemistry, Vol. 67,No. 1, January 7, 7995
61
i
Table 1. Compounds Determined In Indivldual PC12 Cells (fmol)
1
2
3
cell 4
5
6
meanl-~fSEM
DA
7.3
4.1
0.49
3.1
0.92
1.2
Glu
1.4 8.2 2.7 0.035 0.22 0.73
2.9 f 1.1 0.32 f 0.17 5.1 f 1.5b 4.1 f 1.8 3.1 f 1.1 0.18 ?c 0.11
compd
1.05 0.53 0.005 ND4 ND4 0.32 Ala Taur 2.8 8.5 1.2 4.3 off-scale 9.0 12.9 Gly 2.2 3.3 1.1 2.8 2.5 Asp
0.71 3.6 0,090 ND4
2.0
ND4
4 Not detected. Amount cell - amount blank 5 0. A value of 0 is used for calculation of mean and SEM. Because taurine peak is offscale for cell, n = 5.
taurine to glycine is much higher in cell experiments compared to background experiments in all cases, and the ratio of glutamic acid to aspartic acid is typcially higher in the cell experiments relative to background experiments. Despite the presence of peaks due to substances found in the cell medium, electropherograms from single PC12 cells are qualitatively easy to distinguish from background separations. Quantitation of compounds in single PC12 cells is possible if compounds in the cell medium injected with the cell are quantitated. In addition to the peaks for analytes that have been identified and quantitated in PC12 cells, there are a number of peaks that have not yet been identified. The unidenlitied peak eluting at just over 23 min in Figure 1 is present in every cell electropherogram but has not yet been identifed. The peak at just under 13 min in Figure 2 is found in each cell with the exception of cell 3. A third unidentified peak has been found in cells 1, 4, and 6 and has a migration time near 21 min. A number of unidentified peaks are shown in Figure 2, but these peaks will be difficult to identify with the separation conditions used here. At least 13 amino acids have been found to elute in the region of the electropherogram between dopamine and alanine. A large number of NDAderivatized amino acids have been resolved by other researchers using micellar electrokinetic capillary ~hromatography,~~ and this separation strategy could be used in the future to resolve these peaks. One interesting feature of the electropherogram in Figure 1 is the peak at 16.3 min. This peak is present in the electropherograms from all six cells, and it has an average electrophoretic mobility of (-1.74 f 0.01) x 10-4 cmZ/Vs. This peak is absent in all of the control injections. Evidently the peak is caused by something injected with the PC12 cells, as it is present only in electropherograms from the injection of single cells. The peak is also extremely narrow and exhibits an average separation efficiency of (9.5 f 3.6) x lo6 theoretical plates and an average base width of 1.9 f 0.4 s. The extreme narrowness of the peak indicates that it is due to either a large labeled species with a very low diffusion coefficient or some source of scattered light. If this peak is a result of either air bubbles from the cell injection or precipitates from the NDA reaction, it should be present in the electropherograms from the control experiments. It also seems unlikely that a peak from a source like this would have such a consistent electrophoretic mobility over six cell experiments. It is possible that this peak represents a small group of (37) Ueda, T.; Mitchell, R; Kitamura, F.; Metcalf, T.; Kuwana, T.; Nakamoto, A J. Chromatogr. 1992, 593, 265-274.
62 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
I .
10
13
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19
21
25
211
Time (min)
Figure 3. Electropherogram of NDA-derivatized lysate from one dish of PC12 cells. Peak quantities determined are as follow: ValTyr-Val, intemal standard, 3 x 10-5 M; norepinephrine (NE), 18 amol, 4.9 x lo-* M; dopamine (DA), 550 amol, 1.6 x M; Ala, 320 amol, 1.0 x 10-6 M; taurine (T), 3.2 fmol, 1.1 x M; Gly, 450 M; Glu, 1.3fmo1, 6.7 x M; Asp, 140 amol, 7.7 amol, 1.5 x 10-7 M.
specific organelles from the lysed PC12 cell. However, we have not been able to determine the exact identity, as yet, of the species producing this peak. Quantitative Comparison of Amines in PC12 Cells. The dopamine content determined in the six PC12 cells is almost identical to that originally reported by Greene and Tischler (2.9 f 0.5 fmol/cell).31 However, norepinephrine (detection limit, 0.01 fmol) was detected in only one cell in this study (cell 2,0.92 fmol), and Greene and Xschler reported norepinephrine levels of 1.0 f 0.2 fmol/cell?l Figure 3 is an electropherogram of the lysate from an entire culture dish of PC12 cells from the same set used in these experiments. The relative levels of dopamine to norepinephrine determined in the lysate is 31:l. This ratio is similar to that found by summing the amounts of dopamine and norepinephrine for cells 1-6 (19:l). This is within the range of values found in other studies for the ratio of dopamine to norepinephrine in PC12 subclones?* In addition to changes due to clonal variability,%catecholamine levels in PC12 cells have been shown to vary significantly as a function of cell cell density,40 and treatment with nerve growth factor.41 It seems likely that catecholamine levels in a single PC12 cell in a culture dish will vary from other cells in the same dish due to variability of the local environment and cell development. The PC12 cells injected in this study range from approximately 15 to 25 pm in diameter and were at least 2-3 cell diameters away from their nearest neighbors. The majority of the cells in the dishes are found in clumps of many cells, as reported in earlier studies.31 Most of the characterization of PC12 cells, to date, has involved the study of catecholamines and proteins involved in catecholamine synthesis and metabolism. Little has been reported about the amino acid content of these ~ e l l s . 3One ~ study has reported levels of several amino acids in PC12 cells, including alanine, glutamic acid, and aspartic acid.42 These reults were obtained by analyzing entire culture dishes using HPLC with electrochemi(38) Koike, T.; Takashima, A J. Neurochem. 1984,42, 1472-1475. (39) Takashma, A; Koike, T.Biochim. Biophys. Acta 1985, 847, 101-107. (40) Badoyannis, H. C.; Sharma, S. C.; Sabban, E. L. Mol. Brain Res. 1991, 11, 79-87. (41) Greene, L. A; Rein, G. J. Neumchem. 1978, 30, 549-555. (42) Zielke, H. R; Tildon, J. T.; Kauffman, F.C.; Baab, P.J. J. Neurosci. Res. 1989,22,418-424.
Special Considerationsfor On-Column Derivatization of Single Cells. The narrow-bore capillary used in these experi-
in
13
16
19
22
25
28
Time (min)
Figure 4. Electropherogram of a standard solution of amino acids and catecholamines. Injected amounts are as follow: Arg, 1.O x M, 5.2 fmol; Val-Tyr-Val (internal standard), 3.0 x M, 1.2 fmol; NE, 1.0 x M, 3.7 fmol; DA, 5.0 x 10-5 M, 18 fmol; Trp, 1.0 x M, 3.5 fmol; Ala, 1 .O x M, 3.1 fmol; taurine (T), 1.0 x M, 3.0 fmol; Gly, 1.0 x M, 3.0 fmol; dihydroxyphenylalanine M, 2.4 fmol; y-Glu-Gly (internal standard), 1.O x (DOPA), 1.O x M, 2.2 fmol; Glu, 1.0 x M, 1.9 fmol; Asp, 1.0 x M, 1.8 fmol. Injection was 5 s at 10 kV (520 pL). All other conditions are as described in the Experimental Section.
cal detection and precolumn derivatization with OPA. Amino acid levels were reported in nanomoles per milligram of cell protein;42 however, the relative amounts of three amino acids in the PC12 cells can be compared to our results. Zeilke et al.“ reported values of 47.1 f 1.1,88.1 & 3.8, and 30.2 f 1.9 nmol/mg protein for alanine, glutamic acid, and aspartic acid, respectively. The most striking difference between our results and those of Zeilke et al.42 appears to be for glutamic acid levels. In the single-cell experiments reported here, the ratios of glutamic acid to alanine and to aspartic acid are 9.7:l and 179, respectively. The data from Zeilke et al.,43obtained from whole culture dishes, give corresponding ratios of 1.9:l and 2.9:l. The separation of NDA-derivatized lysate from the PC12 cell cultures used here (Figure 3) gives ratios of glutamic acid to alanine and to aspartic acid of 4.1:l and 9.3:1, respectively. Glutamic acid levels appear to be considerably higher in our experiments compared to those of Zeilke et a1.,42 but it is unclear whether this is a result of the analytical methods used in each study or due to variations in PC12 cell cultures. Effect of Cell Injection and Lysing on Separation Efficiency. Figure 4 shows an electropherogram of a standard solution of NDA-derivatized amino acids and catecholamines. Comparison of separationsof standard solutions with single-PC12cell separations have been used to examine the effect of injection of the derivatization reagent and the 10.0 min reaction time on the separation. Examination of the peaks for taurine and glycine in Figures 1 and 4 shows that the separation efficiency has decreased somewhat in the cell experiments. In all six cell experiments, these two peaks were less resolved compared to separationsof standards. Separation efficiencies for identified cell analytes are still excellent in the cell experiments, ranging from 330 000 theoretical plates for aspartic acid to 540 000 theoretical plates for alanine. Separation efficiencies for these compounds are somewhat higher in separations of standards, with glutamic acid showing the largest relative increase in efficiency (23%). (43) de Montigny, P.; Stobaugh, J. F.; Givens, R S.; Carlson, R G.; Srinivasachar, IC;Stemson, L. A; Higuchi, T.Anal. Chem. 1987,59,1096-1101.
ments is critical for successful oncolumn derivatization and capillary electrophoreticanalysis of single cells. Use of an etched capillary has been shown to be not necessary for injection of a single cell into a capillary.12 However, it has been found that use of a capillary that has an etched tip with an outer diameter that is similar in size to the diameter of the cell greatly reduces the difficulty in dislogding the cell from the bottom of the culture dish without rupturing it. Also, maneuvering the capillary to a position near the cell, where the cell will be drawn in by electroosmotic flow, is greatly simplified with a small capillary tip. Since injected cells adhere very easily to the surface of the capillary, the inner conical shape of the injector allows the cell to be drawn further into the capillary before adhering to the capillary wall. The use of an etched tip allows smaller capillaries to be more easily used for cell injection, and lower potentials can be applied to inject the cell. These two factors are important in order to reduce dilution of the contents of the cell during derivatization and in order to reduce the amount of cell medium injected with the cell. The injection end of the capillary serves as a reaction chamber in on-column derivatization of single cells, and the walls of the capillary limit dilution of the reagent and the cell contents during derivatization by restricting diffusion to the longitudinal axis of the capillary. Assuming a spherical geometry, a cell with a diameter of 20 pm will have a volume of 4.2 pL. During a 10.0min reaction time, the root mean square diffusion distance (1 = ( 2 D t ) 9 for a compound with a diffusion coefficient of 5 x cm2/s is 800 pm. As diffusion can only occur down the bore of the capillary, this leads to a total diffusion distance of 1600 pm along the length of the capillary. The volume over this distance for the 17-pm4.d. capillary used in these experiments is 360 pL. The actual time that the contents of the cell have to diffuse away from the cell will be slightly less than 10.0 min because of the iinite amount of time required for the cell to lyse.13J7The reagent injected over the cell (30 s at 2 kV, 27Wpm plug, 600 pL) will also be diluted by diffusion during the lysing and reaction time. The contents of a 2@pm-diametercell are diluted by a factor of approximately 100 during on-column derivatization, but this method represents a signiticant reduction in dilution compared to derivatization in microvials, where final volumes ranged from 20 to 30 nL.6J5,27The use of electrokinetic injection to manipulate picoliter volumes of reagent is critical to reducing the volume in which the derivatization of cellular components takes place. The composition of the reagent buffer is extremely important for on-column derivahtion of single cells. One reagent solution must be able to perform two distinct tasks, lysing the cell and derivatizing the contents of the cell. One can envision using separate injections of two different solutions that are incompatible with each other to lyse the cell and then derivatize its contents. However, the intact cell structure adhering to the capillary wall prevents the cellular contents from migrating down the capillary ahead of the injection of derivatizing reagent. If the cell is lysed prior to injection of derivatiziig reagent, the derivatizing reagent can only electrophoretically mix with the contents of the cell if all components of the derivatization reagent migrate faster than the cell contents and if the reagent components do not separate from each other, The reagent used in this work contains digitonin (lysing agent, 50 pM),NDA and NaCN (derivatizing agent, 2.5 mM each) in the separation buffer (100 mM borate, pH 9.5). The Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
63
buffer also contains 5%ethanol and 5%acetonitrile, which should assist cell lysing. This mixture has been injected separately to determine whether it produces any peaks that interfere with the determination of analytes in single cells. Only one small peak is observed in these experiments. Apparently, an amine impurity is present and reacts with the NDMCN- reagent. This peak is due to a neutral species and has been used as a marker for electroosmotic flow in these experiments. An important feature of the NDNCN- reagent is that it is not fluorescent, and only the products of the derivatization reaction fluoresce strongly.43 Use of reagents that fluoresce would necessitate the separation of a large quantity of reagent from analytes of interest, complicating the method. The reaction rate of the derivatization reagent used for oncolumn derivatization is also very important. Use of a reagent that reacts very slowly with the analytes of interest in a cell will result in unacceptable dilution of the cellular contents due to diffusionduring derivatization and will result in decreased resolution for the electrophoretic separation by the same process. The reaction of NDNCN- with most primary amines is reasonably rapid,a and the reaction time under the conditions used in these experiments has been examined using standard solutions of several of the analytes determined in single PC12 cells and the internal standards used in these experiments. The compounds have been derivatized at concentrations within an order of magnitude of the detection limit of each compound in order to examine the reaction at the slowest rates expected to be encountered in cell experiment^.^^ The derivatization of each of the compounds tested (Val-%-Val, y-Glu-Gly, NE, DA, Gly, and Asp) has been determined to be at least 90%completed by 7.0 min at low analyte concentrations. The derivatization of the analytes quantitated in single cells should be essentially complete in the 10.0-min reaction time used in cell experiments. The pH of the contents of an injected cell and the buffer which is injected with the cell (pH7.4) could potentially affect the kinetics of the derivatization reaction, as they are below the optimal pH for derivatization of amino acids with NDA/CN-.43 However, the injected cell and the small zone of cell medium injected are surrounded by the separation buffer and reagent solution, which are both buffered at pH 9.5. The pH of the cell and the cell
64 Analytical Chemistry, Vol. 67, No. I , January I , I995
medium should equilibrate very rapidly to pH 9.5 due to proton dfision, and the derivatization reaction should not be slowed significantly. Any buffers bordering the zone of sample and derivatization reagent during the reaction should be buffered at a pH that is optimal for the derivatization reaction. CONCLUSIONS Quantitative analysis of single mammalian cells using oncolumn derivatization and capillary electrophoreticseparation with LIF detection has been demonstrated. The method uses the injection end of the capillary as a subnanoliter reaction chamber, where electromigration is used to inject a single cell and to inject reagents to lyse and derivatize the cell. Dilution of the cellular contents is reduced by more than an order of magnitude relative to single-cell derivatization using microvials.6J5827 This method has been used for derivatization and capillary electrophoretic analysis of individual PC12 cells in culture. Femtomole to attomole levels of dopamine and five amino acids have been quantitatively determined in these cells, and several peaks in the cell electropherograms remain to be identitied and quantitated. Accurate quantitation of substances in single cells requires the use of two internal standards-one to correct for both components found in the cell medium and variability in the volume of medium injected when acquiring a single cell, and the other to correct for derivatization reaction and injection procedures. It should be possible to use this method for quantitative analysis of the contents of other types of cells injected in vitro and in vivo. ACKNOWLEDGMENT The authors would like to thank Ta Kung Chen and Susan Zerby for maintaining the PC12 cell cultures used in this work. This research was supported, in part, by a grant from the National Institutes of Health. Andrew G. Ewing is a Camille and Henry Dreyfus Teacher-Scholar. Received for review Ausust 10, 1994. Accepted October
26, 1994.@ AC940792D Abstract published in Advance ACS Abstracts, December 1, 1994.
