Electrically Assisted Sampling across Membranes with

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Electrically Assisted Sampling across Membranes with Electrophoresis in Nanometer Inner Diameter Capillaries Lori A. Woods, Parul U. Gandhi, and Andrew G. Ewing*

Department of Chemistry, 152 Davey Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802

A nondestructive method for sampling from ultrasmall environments has been developed utilizing electrophoresis in nanometer inner diameter capillaries and etched electrochemical detection. The desire to study increasingly smaller biological environments such as mammalian cells has led to the need for capillary electrophoresis techniques with subpicoliter volume sampling capabilities. This sampling technique involves the fabrication of a microinjector at the tip of a 770-nm-inner diameter capillary and the use of electroporation for insertion through the membrane. Separations of catecholamines sampled from the interior of intact liposomes have been achieved. A separation of a cytoplasmic sample taken from an intact mammalian cell has also been obtained. Capillary electrophoresis (CE) has been established as a powerful tool for the study of small biological environments such as single cells. It provides fast and efficient separations of smallvolume samples ranging from nanoliters to picoliters. In addition, subpicoliter samples have been analyzed with the use of CE in nanometer inner diameter (i.d.) capillaries.1-4 These small-volume capabilities have allowed the use of CE for sampling the contents of an entire cell, as well as sampling only a subcellular portion of a large cell. However, the injection of a sample from a cell is often challenging because it requires careful positioning and manipulation of both the capillary and the cell. Therefore, the development of precise sample injection techniques has been crucial to the application of CE to the investigation of ultrasmall environments. Several novel injection methods for single-cell analysis with CE have been developed to minimize cell perturbation during injection.5-10 A multipurpose injector for cell sampling and chemical cytometry using CE has been designed.7,10 To eliminate the * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (814) 863-8081. (1) Wei, G.; Gordon, M. J.; Shear, J. B. Anal. Chem. 1998, 70, 3470-3475. (2) Gostkowski, W.; Shear, J. B. Anal. Biochem. 1998, 260, 244-250. (3) Woods, L. A.; Roddy, T. P.; Paxon, T. L.; Ewing, A. G. Anal. Chem. 2001, 73, 3687-3690. (4) Woods, L. A.; Ewing, A. G. Chem. Phys. Chem. 2003, 4, 207-211. (5) Sims, C. E.; Meredith, G. D.; Krasieva, T. B.; Berns, M. W.; Tromberg, B. J.; Allbritton, N. L. Anal. Chem. 1998, 70, 4570-4577. (6) Meredith, G. D.; Sims, C. E.; Soughayer, J. S.; Allbritton, N. L. Nat. Biotechnol. 2000, 18, 309-312. (7) Krylov, S. N.; Starke, D. A.; Arriaga, E. A.; Zhang, Z. R.; Chan, N. W. C.; Palcic, M. M.; Dovichi, N. J. Anal. Chem. 2000, 72, 872-877. (8) Li, H.; Sims, C. E.; Wu, H. Y.; Allbritton, N. L. Anal. Chem. 2001, 73, 46254631. 10.1021/ac048589y CCC: $30.25 Published on Web 02/10/2005

© 2005 American Chemical Society

challenge of removing a cell from its culture substrate, a lasermicropipet system has been developed to lyse a cell before its injection into the capillary.5,6,8 A similar method using a single electrical pulse for rapid cell lysis also has been utilized.9 However, these techniques have been designed and used for the analysis of whole cells. To sample only a portion of an intact cell with CE, the cell membrane must be penetrated to gain access to the cellular contents. The elasticity of the cell membrane makes penetration by mechanical means extremely difficult.11 Furthermore, the mechanical force applied to the cell membrane can cause permanent damage to the cell or rupture the cell membrane.11 Electroporation has been investigated as an alternative method of gaining access to the interior of a cell.12-14 Electroporation is the formation of transient pores in the cell membrane that results from exposure to a pulse of high voltage that destabilizes the membrane potential.12 Electric field strengths of 1-1.5 kV/cm applied for several microseconds to several milliseconds are sufficient to cause pore formation in the membrane of a 10-µmdiameter cell.12 Overall, these pores range in size from 1 to 240 nm.13 The formation of pores facilitates penetration of the cell membrane and allows access to the cellular contents.14 Electroporation has been used to introduce materials into the interior of ultrasmall environments including liposomes and cells.13,14 In this paper, a method for relatively nondestructive sampling from ultrasmall environments using electrophoresis in nanometer i.d. capillaries and etched electrochemical detection is presented. This new sampling technique involves the fabrication of a microinjector at the tip of a 770-nm-i.d. capillary and the use of electroporation for insertion. These methods allow careful positioning of the capillary and minimize damage to the sample. Liposomes are used as a model system for future work with single cells, and separations of catecholamines sampled from the interior of intact liposomes have been achieved. As little as 2% of the total (9) Han, F.; Wang, Y.; Sims, C. E.; Bachman, M.; Cheng, R.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2003, 75, 3688-3696. (10) Krylov, S. N.; Dovichi, N. J. Electrophoresis 2000, 21, 767-773. (11) Chien, J. B.; Wallingford, R. A.; Ewing, A. G. J. Neurochem. 1990, 54, 633638. (12) Lundqvist, J. A.; Sahlin, F.; Aberg, M. A. I.; Stromberg, A.; Eriksson, P. S.; Orwar, O. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10356-10360. (13) Karlsson, M.; Nolkrantz, K.; Davidson, M. J.; Stromberg, A.; Ryttsen, F.; Akerman, B.; Orwar, O. Anal. Chem. 2000, 72, 5857-5862. (14) Nolkrantz, K.; Farre, C.; Brederlau, A.; Karlsson, R. I. D.; Brennan, C.; Eriksson, P. S.; Weber, S. G.; Sandberg, M.; Orwar, O. Anal. Chem. 2001, 73, 4469-4477.

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volume of a liposome has been sampled and analyzed with this technique. Furthermore, in a preliminary demonstration, a separation of a subcellular sample taken from an intact mammalian cell has been carried out with this technique. EXPERIMENTAL SECTION Reagents. Dopamine (DA), catechol (CAT), isoproternol (ISO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), D-(+)-glucose, and Trizma base were obtained from Sigma (St. Louis, MO). A 48% aqueous solution of hydrofluoric acid was obtained from Aldrich (Milwaukee, WI). All chemicals were used as received. The separation buffer used was 50 mM TES containing 2% 1-propanol adjusted to pH 7.2 with sodium hydroxide. All standards were prepared as 100 mM stock solutions in 0.1 M perchloric acid and were diluted to the desired concentration with the separation buffer or 30 mM phosphate buffer adjusted to pH 7.8 with sulfuric acid. Physiological saline was prepared as previously described and adjusted to a pH 7.7 with sodium hydroxide.15 Liposome Preparation. Soybean Polar Extract lipids were obtained from Avanti Polar Lipids, Inc. (Alasbaster, AL). These lipids primarily consisted of phophatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and phosphatic acid. The procedure for liposome preparation has been described previously.16 Briefly, a 10 mg/mL mixture of soybean lipids in chloroform was prepared. A 300-µL aliquot of this solution was placed on a rotary evaporator for 6 h. Phosphate buffer was added to the remaining lipid film to a final lipid concentration of 1 mg/mL. The lipid film was stored overnight at 4 °C to allow it to swell. In preparation for use, the lipid film was sonicated in an ice bath for 10 min. A 5-µL aliquot of the mixture was placed on a plastic microscope slide and then held in a desiccator under vacuum for ∼30 min. To rehydrate the liposomes, 10 µL of a solution containing CAT in phosphate buffer was placed over the lipid mixture on the plastic slide. After 10 min, 100 µL of phosphate buffer was added. Cell Culture. Rat pheochromocytoma (PC12) cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as previously described.17 Briefly, the cells were grown on 35-mm culture dishes coated with mouse collagen IV in RPMI-1640 medium supplemented with horse serum, fetal bovine serum, and penicillin-streptomycin. The cells were kept in a 7% CO2 atmosphere at 37 °C. The cells were subcultured approximately every 7-9 days and then used for experiments within 7 days of subculturing. Prior to performing an experiment, the cell medium in the culture dishes was removed and replaced with physiological saline. Apparatus. A CE system with end-column amperometric detection was utilized. This system has been previously described.4,18,19 Briefly, 90-95 cm of fused-silica capillary with an outer diameter of 150 µm and i.d. of 770 ( 40 nm (Polymicro Technologies, Phoenix, AZ) was employed. The i.d. of the (15) Colliver, T. L.; Pyott, S. J.; Achalabun, M.; Ewing, A. G. J. Neurosci. 2000, 20, 5276-5282. (16) Cans, A. S.; Wittenberg, N.; Karlsson, R.; Sombers, L.; Karlsson, M.; Orwar, O.; Ewing, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 400-404. (17) Kozminski, K. D.; Gutman, D. A.; Davila, V.; Sulzer, D.; Ewing, A. G. Anal. Chem. 1998, 70, 3123-3130. (18) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581. (19) Huang, Z.; Sloss, S.; Ewing A. G. Anal. Chem. 1991, 62, 189-192.

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capillaries was confirmed using scanning electron microscopy (SEM) and is reported as the mean and standard deviation of four measurements. Amperometric detection was performed using a two-electrode configuration. A 5-µm-diameter carbon fiber (Amoco Performance Products, Greenville, SC) was employed as a working electrode. The carbon fiber microelectrode was positioned inside the end of the capillary using a micromanipulator. Electrochemical detection was performed at 0.7 mV versus an Ag/AgCl reference electrode. The detection system was enclosed in a copper mesh Faraday cage to minimize external noise. Injections were performed electrokinetically, and injection volumes were calculated based on electroosmotic flow measured with a neutral marker. Procedures. The ultrasmall capillaries were filled by using a liquid chromatography pump (Scientific Systems, State College, PA). This procedure has been previously described.3 The capillaries were filled with a mixture of methanol and doubly distilled water at pressures of 4000-5000 psi. A microinjector was fabricated at one end of the capillary. Approximately 1 cm of the polyimide coating was removed from the capillary to expose the fused silica. The exposed portion of the capillary was placed in hydrofluoric acid. After 10 min, the capillary was repositioned so that ∼2 mm of the exposed portion of the capillary was no longer in contact with the hydrofluoric acid. Exposure to hydrofluoric acid continued for an additional 35 min. The capillary was placed in a sodium bicarbonate solution to neutralize the acid and then washed with water. Using a scalpel, the tip of the exposed portion of the capillary was trimmed to maintain the nanometer i.d. of the capillary. The exposed portion of the capillary was placed in hydrofluoric acid for an additional 1-10 min to create a tapered microinjector with a tip outer diameter of 2.5 µm. To increase the stability of the microinjector, a 5-cm piece of 0.75-mm-i.d. glass capillary (Sutter Instruments Co., Novato, CA) was used as a holder. The capillary was threaded through the glass capillary and then glued in place so that only the microinjector protruded from the holder. Approximately 2 mm of the polyimide coating was removed from the other end of the capillary to expose the fused silica. The exposed portion of the capillary was placed in hydrofluoric acid for 5 min. The i.d. of the capillary was etched open with hydrofluoric acid to accommodate the carbon fiber microelectrode. After the etching was complete, the exposed portion of the capillary was placed in a sodium bicarbonate solution to neutralize the acid and then washed with water. The carbon fiber microelectrode was etched in an acetylene flame. It was positioned in the flame at a 45° angle for 1-2 s. This created a microelectrode with a 2.5-µm-diameter conical tip and an electroactive length of 200-400 µm. Injections of samples taken from the interior of a liposome or cell were performed with the aid of electroporation. A platinum wire was used as a counter electrode and placed in contact with the solution containing the liposomes or cells. With the use of an Olympus CK30 microscope (Melville, NY) and a micromanipulator, a microinjector fabricated at the end of an ultrasmall capillary was positioned against the liposome or cell with enough force to cause a slight indentation in the membrane. A voltage of either 2 or 3 kV was applied for between 2 and 6 s. This applied voltage induces pore formation in the membrane that eases the penetra-

Figure 1. Schematic of the injection procedure. (a) A microinjector fabricated at the end of an ultrasmall capillary is positioned against the liposome with enough force to cause a slight indentation in the liposome. (b) A voltage of 2-3 kV is applied for 2-6 s. (c) This applied voltage induces pore formation in the liposomal membrane that eases the penetration of the liposome with the microinjector and causes the electrokinetic injection of a sample from the interior of the liposome into the capillary.

tion of the liposome or cell with the microinjector and causes the electrokinetic injection of a sample from the interior of the liposome or cell. This procedure is outlined in Figure 1. All injections were performed on a Benchtop Vibration Isolation System (Newport, Irvine, CA). Safety Considerations. A safety interlock box was used to protect the user from high voltage. Hydrofluoric acid can cause severe burns and must be used with extreme care. It should be neutralized with sodium bicarbonate prior to disposal. RESULTS AND DISCUSSION Microinjector for CE in Nanometer i.d. Capillaries. A procedure has been developed to fabricate a microinjector on the end of a 770-nm-i.d. capillary using hydrofluoric acid. The microinjector tapers from an outer diameter of 150 to 2.5 µm at the tip. To confirm the shape and dimensions of the microinjector, SEM images were obtained. A SEM image of a microinjector at a magnification of 50 is shown in Figure 2a, and a SEM image of the tip of a microinjector at a magnification of 7500 is shown in Figure 2b. This image indicates that the i.d. of the capillary at the tip of a microinjector is slightly larger than 770 nm. The slight change in the i.d. of the capillary at its tip will not significantly affect the volume injected into the capillary for analysis, which is determined by the potential field across the entire capillary. In this initial application, the performance of the sampling technique was investigated. Using a microinjector on a 770-nmi.d. capillary, a separation of a 6-pL sample of DA and CAT has been carried out and is shown in Figure 3. As expected, DA elutes first followed by the neutral, CAT. The peak efficiencies for both DA and CAT in this separation have been calculated to be ∼100 000. These peak efficiencies are comparable to those achieved for DA and CAT with etched electrochemical detection in a 770-nm-i.d. capillary without a microinjector.4 Sampling from a Liposome with Electrophoresis in a Nanometer i.d. Capillary. Direct sampling of cell cytoplasm from a cell in culture is extremely challenging. Mammalian cells in culture sit on the Petri dish resembling a swollen disk. Forcing a micropipet or the etched end of a capillary into the cell results in 100% failure in our hands. This is also true for injections from liposomes adhered on surfaces. We were not able to do any successful mechanical insertions/sampling from either liposomes or cells. For liposomes under mechanical force, in 90% of the trials, the liposome would detach from the slide, pool of lipids, or multilayer liposome that was holding it in place and float away. The other 10% of trials, the lipsome wall would smear across the injector and then the liposome would collapse. For cells under

Figure 2. SEM images of a microinjector fabricated at the end of a 770-nm-i.d. capillary showing its size and integrity. (a) Microinjector at a magnification of 50. Scale bar represents 500 µm. (b) Tip of microinjector at a magnification of 7500. Scale bar represents 1 µm.

Figure 3. Electropherogram obtained using a microinjector etched at the end of 770-nm-i.d. capillary. Conditions: 10-s injection at 10 kV of a 1 mM solution of DA and CAT; 25-kV separation potential; 50 mM TES (pH 7.2) separation buffer with 2% 1-propanol added. The total injection volume in this separation was 6 pL.

mechanical force from the injector, the cell would slide across the dish usually followed by the capillary tip being destroyed on the plate bottom. Using electroinsertion, injection into etched capillaries was uniformly successful. An injection and detection of 1 mM CAT sampled from the interior of a 10-µm-diameter liposome is shown in Figure 4a and is compared to an injection of the CAT solution used to fill the liposome, which is shown in Figure 4b. The CAT eluted from the capillary at 15.2 and 14.9 min, respectively. This slight variation in the migration time of CAT is most likely the result of the injection of some of the lipid material and possibly the interaction of the capillary tip with the membrane. To further confirm that CAT was being sampled from the interior of the liposome and not from material that had leaked out, a sample was also injected Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Table 1. Relationship between the Percentages of the Total Liposome Volume Injected and normalized Peak Area for CATa total liposome vol (pL)

% of total liposome vol injected

normalized peak area for CAT

10.9 40.2 41.9 2.8 0.5

2 3 6 12 33

0.96 0.91 0.93 0.76 1.10

a Injections were made from the interior of liposomes using a microinjector etched on the end of a 770-nm-i.d. capillary. Each percentage of the total liposome volume injected and its corresponding normalized peak areas was obtained from a separation of CAT sampled from the interior of a different liposome. The liposome volumes were calculated by assuming a spherical shape. The normalized peak areas were calculated as the ratio of the peak area obtained from the separation of sample taken from each liposome divided by the peak area obtained from a separation of a standard CAT solution performed on the same capillary.

Figure 4. Comparison of the electropherograms obtained using (a) a 1 mM solution of CAT in phosphate buffer (pH 7.8) injected from the interior of a 10-µm-diameter liposome, (b) the same 1 mM solution of CAT in phosphate buffer injected from a standard vial, and (c) a control injection from a pool of phosphate buffer containing liposomes which were filled with a 1 mM solution of CAT in phosphate buffer. These separations were performed using a 770-nm-i.d. capillary, a 3-s injection at 3 kV, and the same conditions as in Figure 3.

from the pool of phosphate buffer containing the liposomes (Figure 4c). A peak with a migration time comparable to that of CAT was not observed. Another concern with the utilization of a novel injection technique is the ability to obtain quantative information. The sampling technique has been calibrated using separations of three different amounts of CAT taken from the interior of liposomes containing different concentrations of catechol (2, 1, and 0.5 mM). Each injection was carried out twice on a liposome, and the average amounts and corresponding normalized peak areas for CAT were 531, 295, and 175 amol injected. The data have been normalized to injections in standard solution for each capillary. The resulting calibration plot is linear over this narrow range with a correlation coefficient for the amount of CAT of 0.98, thus suggesting that this technique can be used to obtain quantitative information. The relationship between normalized peak area and the percentage of the total liposome volume injected also has beenexamined. Injections of five different percentages of total liposome volume were performed. The percentages and corre1822 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Figure 5. Comparison of the electropherograms obtained using (a) a 3 mM solution of DA, ISO, and CAT in phosphate buffer (pH 7.8) injected from the interior of a 29-µm-diameter liposome and (b) the same 3 mM solution of DA, ISO, and CAT in phosphate buffer used to fill the liposome injected from a vial. Separation conditions were the same as Figure 4.

sponding normalized peak areas for CAT are shown in Table 1. All five normalized peak areas fluctuate around a value of 1.0 regardless of the percentage of the total liposome volume injected. This lack of correlation indicates that the percentage of the total liposome volume injected does not affect the quantitative information that can be obtained with this technique. After characterizing the sampling technique, a multicomponent separation of a sample taken from the interior of a liposome with a diameter of 29 µm was accomplished (Figure 5a). The cationic species, DA and ISO, elute first and are resolved in less than 13 min. They are followed by the elution of the neutral, CAT at ∼15 min. For comparison, a separation of the multicomponent solution

Figure 6. Electropherogram of a 280-fL sample injected from a 20µm-diameter PC12 cell obtained using a 770-nm-i.d. capillary, a 5-s injection at 3 kV, a 25-kV separation potential, and a 50 mM TES (pH 7.2) separation buffer with 2% 1-propanol.

used to fill the liposome is shown in Figure 5b. This separation demonstrates that a more complex analysis can be carried out using this technique. Subcelluar Sampling from a Mammalian Cell with Electrophoresis in a Nanometer i.d. Capillary. A preliminary injection from an intact 20-µm-diameter PC12 cell is shown in Figure 6. The injection volume for this separation was 280 fL. Several peaks were observed in the electropherogram including three major peaks at 15.8, 16.7, and 17.6 min. PC12 cells are known to contain catecholamines and their metabolites, but no attempt was made to identify the peaks in the separation of the subcellular sample.20 This represents the first direct cytoplasmic sampling from an intact mammalian cell using CE and, although difficult, promises to open a new level of small-volume sampling in cell analysis. (20) Clark, R. A.; Ewing, A. G. Mol. Neurobiol. 1997, 15, 1-16.

CONCLUSIONS A method for relatively nondestructive sampling from ultrasmall environments using electrophoresis in nanometer i.d. capillaries and etched electrochemical detection has been developed. This new sampling technique involves the use of electroporation to insert a microinjector across a model or cell membrane. The method described allows careful positioning of the capillary with minimal damage to the sample. Separations of catecholamines sampled from the interior of intact liposomes have been achieved and can be used to obtain quantative information. As little as 2% of the total volume of a liposome has been sampled and analyzed using this method. This new sampling technique provides the small-volume capabilities and high sensitivity, as well as gentle injection process, necessary for subcellular sampling from single mammalian cells as demonstrated by a preliminary separation of a cytoplasmic sample taken from an intact mammalian cell. This method could be used to determine neurotransmitter and metabolite levels in the cytoplasm of single mammalian cells. This knowledge will provide insight into neurotransmitter storage and synthesis and should be useful in developing and enhancing models of neurotransmitter transport across cell and vesicle membranes. ACKNOWLEDGMENT The authors thank Nate Wittenberg and Dan Eves for preparation of the liposomes used in these experiments. The National Science Foundation and the National Institute of Health supported this work. Received for review September 23, 2004. Accepted December 30, 2004. AC048589Y

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