Anal. Chem. 1998, 70, 3470-3475
Determination of Fluorogen-Labeled Neurotransmitters at the Zeptomole Level Using Two-Photon Excited Fluorescence with Capillary Electrophoresis Jing Wei, Michael L. Gostkowski, Mary Jane Gordon, and Jason B. Shear*
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712
Two-photon excited (2PE) fluorescence detection is demonstrated to be a highly sensitive means for analyzing fluorogen-labeled neurotransmitters fractionated in submicrometer capillary electrophoresis channels. In this approach, fluorescamine-labeled neurotransmitters that have been electrophoretically separated in 620-nm-i.d. capillaries intersect the focused output from a nearinfrared mode-locked titanium-sapphire laser positioned at the capillary outlet. Extremely high peak laser intensities (∼1011-1012 W cm-2) facilitate the nearly simultaneous absorption of two near-IR photons (λex ≈ 780 nm) to excite fluorescamine derivatives ordinarily excited with a single, near-ultraviolet photon (λex ≈ 390 nm). Rapid cycling of analytes through the fluorescent excited state and low background from scatter and out-of-sample luminescence combine to make 2PE fluorescence a highly sensitive approach for detecting minute quantities of neurotransmitters. In these studies, mixtures of the fluorescamine derivatives of dopamine, glycine, and glutamate are fractionated reproducibly in several minutes, with instrumental mass detection limits as low as 13 000 molecules (∼20 zmol). These detection levels are ∼100-fold lower than have been achieved previously for fluorescamine-based assays. Analytes can be derivatized at concentrations equal to the limit of quantitation with no loss in sensitivity; hence, characterization of neuronal samples at the zeptomole level appears feasible, provided that efficient on-column labeling procedures can be implemented. Microcolumn separation techniques have played an increasingly valuable role in the chemical characterization of volumelimited samples, such as those available from biological microenvironments.1-3 The experimentally simplest of the microcolumn techniques, capillary electrophoresis (CE), is capable of characterizing multicomponent samples ranging in volume from nanoliters to femtoliters4 and can be coupled readily to a diverse selection of high-sensitivity detection approaches.5 Measurements per(1) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (2) Lillard, S. J.; Yeung, E. S.; McCloskey, M. A. Anal. Chem. 1996, 68, 28972904. (3) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845.
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formed using end-column amperometry and ion cyclotron resonance mass spectrometry have yielded subattomole (∼500 000 molecules) detection limits for biological components.6,7 Ultrasensitive fluorescence analysis of biological samples fractionated with CE has relied principally on two alternative strategies: (i) laser excitation of native ultraviolet (UV) fluorescence from indoles and tyrosine derivatives (e.g., tryptophan-containing proteins and peptides, serotonin, catecholamines)2,8,9 and (ii) derivatization of a selected chemical moiety, typically followed by laser excitation of visible fluorescence.10-12 Detection based on native UV fluorescence avoids measurement errors associated with sample preparation on the subnanoliter scale; however, only special classes of biological species exhibit significant native fluorescence. None of the primary excitatory or inhibitory neurotransmitters in the mammalian central nervous system (i.e., glutamate, aspartate, glycine, and γ-aminobutyric acid), for example, can be measured by virtue of intrinsic fluorescence properties. In contrast, fluorescence assays based on derivatization with a reagent specific for primary amines have the potential to measure nearly all currently identified neurotransmitters. Low-concentration fluorescence labeling typically is accomplished using fluorogenic reagents.13-16 Unlike highly fluorescent labels based on fluorescein or rhodamine, these reagents are essentially nonfluorescent but form fluorescent adducts after reaction with analytes. Rapid and efficient labeling of analytes (4) Chiu, D. T.; Lillard, S. J.; Scheller, R. H.; Zare, R. N.; Rodriguez-Cruz, S. E.; Williams, E. R.; Orwar, O.; Sandberg, M.; Lundqvist, J. A. Science 1998, 279, 1190-1193. (5) St. Claire, R. L., III Anal. Chem. 1996, 68, 569R-586R. (6) Ewing, A. G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994, 66, 527A537A. (7) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (8) Timperman, A. T.; Oldenburg, K. E.; Sweedler, J. V. Anal. Chem. 1995, 67, 3421-3426. (9) Tong, W.; Yeung, E. S. J. Chromatogr., B 1997, 689, 321-325. (10) Gilman, S. D.; Ewing, A. G. Anal. Chem. 1995, 67, 58-64. (11) Nickerson, B.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 157-168. (12) Shippy, S. A.; Jankowski, J. A.; Sweedler, J. V. Anal. Chim. Acta 1995, 307, 163-171. (13) de Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096-1101. (14) Roth, M. Anal. Chem. 1971, 43, 880-882. (15) Undenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871-872. (16) Liu, J.; Shirota, O.; Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2302-2306. S0003-2700(98)00296-0 CCC: $15.00
© 1998 American Chemical Society Published on Web 07/03/1998
often is accomplished using fluorogenic compounds because a large molar excess of reagent can be used while maintaining a nominally zero-level fluorescence background. Many fluorogenic species now are available, with diverse chemical specificities, reaction kinetics, spectroscopic properties, and photochemical/ thermal stabilities. High-sensitivity analysis of amino acids, peptides, proteins, and aminated sugars can be performed using several different aminespecific fluorogenic labels, including fluorescamine,12 naphthalenedicarboxaldehyde (NDA),10,17 o-phthaldialdehyde (OPA),18 furoylquinolinecarboxaldehyde (FQCA),19 and carboxybenzoylquinolinecarboxaldehyde (CBQCA).16,20 Zare and co-workers recently demonstrated analysis of taurine and peptides in single 1-µmdiameter endocrine vesicles from Aplysia californica using NDA and laser excitation at 457.9 and 442 nm.4 The lowest limits of detection (LODs) for a fluorogenic assay were reported by Dovichi and co-workers using FQCA to label multiple sites on protein molecules.21 Low-zeptomole levels of conalbumin were analyzed using 488-nm excitation and a sheath-flow cuvette to minimize background from laser scatter. An important factor in the conalbumin study was the choice of the reagent FQCA, which is highly reactive to proteins and forms reaction products that can be excited efficiently by the primary line of the argon ion laser. A drawback when using FQCA, however, is the relatively slow reaction rate: 10 min or more is generally required to achieve complete reaction. In addition, preliminary studies suggest that FQCA-based assays for amino acids and monoamine neurotransmitters yield substantially poorer detection limits than can be achieved for proteins.21 Fluorescaminesan amine-reactive fluorogenic reagent developed more than a quarter of a century ago15soffers a unique combination of desirable characteristics as a probe for small quantities of neurotransmitters. First, the fluorescamine reaction with primary amines is notable for its extremely fast kinetics; when used at millimolar reagent concentrations, reactions are typically complete in milliseconds. Rapid or on-line analyses of biological fluidssfor example, the continual sampling of brain diasylate18s are simplified substantially when fast labeling reactions are used. Second, fluorescamine has good reactivity and reasonable fluorescence quantum yields for diverse classes of analytes, including the monoamine and amino acid neurotransmitters, peptides,12 and proteins.22 Some of the fluorogenic reagents are more restricted in applicability. OPA, for example, is a useful probe for amino acids and proteins but forms relatively nonfluorescent adducts with short peptides. Orwar et al. demonstrated that low fluorescence generated from OPA-labeled peptides is caused by poor fluorescence quantum yields (Φf) and large photodestruction quantum yields (Φd).23 (17) de Montigny, P.; Riley, C. M.; Sternson, L. A.; Stobaugh, J. F. J. Pharmaceut. Biomed. Anal. 1990, 8, 419-429. (18) Lada, M. W.; Vickroy, T. W.; Kennedy, R. T. Anal. Chem. 1997, 69, 45604565. (19) Pinto, D. M.; Arriaga, E. A.; Craig, D.; Angelova, J.; Sharma, N.; Ahmadzadeh, H.; Dovichi, N. J. Anal. Chem. 1997, 69, 9, 3015-3021. (20) You, W. W.; Haugland, R. P.; Ryan, D. K.; Haugland, R. P. Anal. Biochem. 1997, 244, 277-282. (21) Dovichi, N. J.; Arriaga, E.; Krylov, S.; Ahmadsadeh, H.; Pinto, D.; Tan, W.; Shang, Z. Single Cell Proteome Mapping. Eleventh International Symposium on High Performance Capillary Electrophoresis, Orlando, FL, Feb 1998. (22) Stein, S.; Moschera, J. Methods Enzymol. 1981, 79, 7-16.
Despite the attributes of fluorescamine as a reagent for neurotransmitters, the inconvenient spectral properties of fluorescamine derivatives has impeded use of this probe in highsensitivity, volume-limited analyses. The highly fluorescent pyrrolinone products formed in the fluorescamine reaction with amines are optimally excited at ∼390 nm, a spectral region difficult to access using reliable, stable laser sources. Sweedler and coworkers demonstrated sensitive detection of fluorescamine-labeled neuropeptides, achieving detection limits of 1.0) microscope objective. The low duty cyle of the femtosecond-pulsed Ti-S laser (∼10-5) maintains a relatively low average intensity, thus avoiding significant sample heating and minimizing background that scales linearly with laser intensity (23) Orwar, O.; Weber, S. G.; Sandberg, M.; Folestad, S.; Tivesten, A.; Sundahl, M. J. Chromatogr., A 1995, 696, 139-148. (24) Shear, J. B.; Brown, E. B.; Webb, W. W. Anal. Chem. 1996, 68, 17781783.
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(e.g., Rayleigh scatter). Moreover, the high repetition rate of the Ti-S laser (∼75 MHz) provides the capability for rapidly cycling chromophores through the excited state. MPE fluorescence has been used previously as a detection approach with chromatography, although the low repetition rate and long pulse widths of the laser sources available for these earlier studies were not conducive to its optimal implementation.25-27 Recently, multiphoton excitation with a Ti-S laser has been adapted for use with capillary electrophoresis, providing a versatile, reproducible, and sensitive strategy for measuring laser dyes28 and various biological species.29 Fluorescence analysis of samples containing spectroscopically distinct biological components (including melatonin and flavin adenine dinucleotide) was accomplished by exciting near-UV and deep-UV chomophores with the combined energy of different numbers of near-IR photons.29 In more recent studies, serotonin and related compounds were quantified after separation in 600-nm-i.d. capillaries using MPE hyperluminescence,30 a process in which intense visible emission is generated from hydroxyindoles as a result of photochemical transformation.31 Using this strategy, mass detection limits for serotonin (∼40 000 molecules) were improved more than 10-fold compared to UV fluorescence assays. In this work, we demonstrate the use of 2PE fluorescence analysis with capillary electrophoresis to measure extremely small quantities of neurotransmitters labeled with fluorescamine. The mass detection limits in these studiessfewer than 15 000 moleculessreveal a strategy that may prove useful in probing fundamental biological chemistry, provided that fluorogenic labeling of minute samples can be accomplished. EXPERIMENTAL SECTION Chemicals and Materials. Fluorescamine was obtained from Molecular Probes (Eugene, OR), and was dissolved in HPLCgrade acetone (g99.5%, EM Science). All other standards and reagents were obtained from Sigma (St. Louis, MO) and were used without further purification. Water was purified using a Barnstead UV water system, and all aqueous solutions were filtered with 0.22-µm cellulose acetate filters (Osmonics, Livermore, CA) to remove particulates that could obstruct the capillary channel. Fused-silica separation capillaries were obtained from Polymicro, Inc. (Phoenix, AZ). Sample Preparation. 5-(2-Carboxyphenyl)-5-hydroxy-3-phenyl-2-pyrrolin-4-one (CPP) derivatives of neurotransmitters were prepared by adding 500 µM fluorescamine in acetone (400 µL) to either mixtures or individual solutions of dopamine, glycine, and glutamate (each at 120 µM) in 10 mM borate buffer (pH 9.1, 200 µL) while rapidly vortexing the neurotransmitter solution. Reaction mixtures were diluted with CE running buffer (see below) before analysis to provide samples nominally composed of 10 µM of the three labeled neurotransmitters. In addition, glycine was (25) Sepaniak, M. J.; Yeung, E. S. Anal. Chem. 1977, 49, 1554-1556. (26) Pfeffer, W. D.; Yeung, E. S. Anal. Chem. 1986, 58, 2103-2105. (27) Nesse, R. J. v. d.; Mank, A. J. G.; Hoornweg, G. P.; Gooijer, C.; Brinkman, U. A. T. Anal. Chem. 1991, 63, 2685-2688. (28) Song, J. M.; Inoue, T.; Kawazumi, H.; Ogawa, T. J. Chromatogr., A 1997, 765, 315-319. (29) Gostkowski, M. L.; McDoniel, J. B.; Wei, J.; Curey, T.; Shear, J. B. J. Am. Chem. Soc. 1998, 120, 18-22. (30) Gostkowski, M. L.; Wei, J.; Shear, J. B. Anal. Biochem., in press. (31) Shear, J. B.; Xu, C.; Webb, W. W. Photochem. Photobiol. 1997, 65, 931936.
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reacted at low concentration (0.75 µM) and was injected directly into the CE capillary without dilution. Laser System. The excitation source for all experiments was a Coherent Mira 900F femtosecond mode-locked Ti-S oscillator pumped by an Innova 310 multiline argon ion laser. The Miras which was operated at 780 nm (fwhm ∼10 nm) for all studiess typically yields sech2 pulses (pulse width 100-150 fs; repetition rate 76 MHz) and has a relative rms power noise of ∼1%. For optimal signal-to-noise ratios in CE measurements, the Mira output was attenuated using a half-wave plate/polarizer pair, providing an average power at the capillary outlet of ∼75 mW. Multiphoton Excitation Capillary Electrophoresis System. Analytes were fractionated in a 620-nm-i.d. capillary (180-µm o.d.; capillary length 20.8 or 24.0 cm) using a separation voltage of 15 kV. The inner diameter of the capillary was determined by imaging the capillary tip with a scanning electron microscope. All samples were injected electrokinetically using a voltage of 2 kV for 3s, and separations were performed using 20 mM Hepes buffer (pH 7.0). After fractionation, labeled neurotransmitters were detected using an end-column approach similar to that described previously.29,30 Safety Considerations: The high voltage inlet for electrophoretic separations should be isolated from users during operation with an interlocked plastic box. In short, a high-NA microscope objective (Zeis Fluar, 1.3 NA oil immersion, infinity-corrected) focuses the Ti-S beam to a diffraction-limited spot characterized by ∼250 nm radial and ∼900 nm axial 1/e2 intensity radii (on the central diffraction spot). This high-intensity focal region (pulse intensities 1011-1012 W cm-2) is positioned at the outlet aperture of the separation capillary using a three-axis translation stage driven by high-resolution differential micrometers (beam propagation is directed along the capillary axis, toward the inlet). Analytes migrating from the capillary into the outlet (cathode) buffer reservoir intersect the focal region as they pass through the outlet aperture. The resultant fluorescence is collected in an epi configuration using the same objective that focuses the excitation light, and fluorescence at wavelengths shorter than 620 nm is reflected at 90° from the beam path using a dichroic mirror (Chroma Technology, 625DCXR). A colored glass filter (Schott Glass, BG18) and 2 cm of 1 M CuSO4 solution are used to isolate fluorescence from laser scatter and other potential sources of background. Signal is measured with a photomultiplier tube (Hammamatsu, HC125-02), connected to a photon counter (Stanford Research Systems, SR400), which transfers fluorescence data through a GPIB interface to a Macintosh running LabView-based software. A diagram of the overall detection system is shown in Figure 1. Visualization of the capillary outlet is accomplished by splitting off and focusing a portion of the Ti-S light onto a thin spot of epoxy on the capillary (within the outlet solution reservoir). The epoxy scatters some light at a small angle relative to the capillary axis, thereby coupling the light into the fused-silica capillary walls so that it propagates to the outlet by total internal reflection. Light radiating from the end of the capillary is collected by the microscope objective, reflected by beam splitter (B2) and imaged onto a CCD video camera (Figure 1). Using this procedure, the outlet aperture of the capillary appears as a dark circle surrounded by a relatively bright “donut” of fused silica. The focal spot formed by the microscope objective is observed in this image as a
Figure 1. Diagram of the end-column two-photon fluorescence detection system for capillary electrophoresis. Abbreviations: Ti-S, titanium-sapphire laser; B1 and B2, beam splitters; D, dichroic mirror; F, filters; obj, microscope objective; cap, separation capillary. Only the last few centimeters of the capillary are shown in this diagram.
Figure 3. Fractionation of a subpicoliter sample containing fluorescamine derivatives of dopamine, glycine, and glutamate. Analytes are labeled off-column at high concentration and are diluted before analysis. Capillary length 20.8 cm.
1.07-µm-diameter polystyrene sphere adsorbed to the fused-silica surface.
Figure 2. A video image of the end of the capillary showing the excitation focal spot aligned with the 620-nm channel aperture (top panel). To illustrate the channel diameter, a scanning electron micrograph is shown with 1.07-µm polystyrene sphere adsorbed to the fused-silica surface (lower panel).
reflection off the fused silica; by translating the capillary, the reflection can be made to (essentially) disappear within the submicrometer channel aperture. After this alignment procedure has been performed, the beam splitter B2 is removed and a separation is performed. In general, alignment is verified between separations, although drift on this time scale is typically small. Figure 2 (top panel) demonstrates a video image of the capillary end with the focal spot positioned at the channel aperture. To better illustrate dimensions, the bottom panel of Figure 2 shows a scanning electron micrograph of the aperture in proximity to a
RESULTS AND DISCUSSION Figure 3 demonstrates the ability of this 2PE fluorescence system to analyze samples containing very small amounts of several derivatized neurotransmitters. A mixture of dopamine, glycine, and glutamate was derivatized at high concentration and was diluted before analysis. Assuming a 100% reaction efficiency, CPP-dopamine, CPP-glycine, and CPP-glutamate all were present in the injected sample at 10 µM. Injection volumes for the three species vary according to their respective mobilities, ranging from ∼310 fL for CPP-dopamine to ∼130 fL for CPP-glutamate. Detection limits (amounts needed to generate signal 3-fold larger than the rms noise) for this separation are calculated to be ∼57 zmol (34 000 molecules) for CPP-dopamine, ∼22 zmol (13 000 molecules) for CPP-glycine, and ∼33 zmol (20 000 molecules) for CPP-glutamate. Concentration detection limits range from 110 nM for CPP-glycine to 250 nM for CPP-glutamate. Although these concentration detection limits are relatively high because of the extremely small sample volumes,32 the mass detection levels are ∼100-fold lower than the best reported results for fluorescaminebased assays with 1PE fluorescence. The linear dynamic range extended from submicromolar concentrations (the limits of quantitation) to several hundred micromolar. The dependence of CPP-neurotransmitter fluorescence peak heights on excitation intensity was determined using a range of laser intensities and was found to scale as I2 for intensities less than ∼50 mW (Figure 4). By plotting the data in a log-log format, the slope of the line reveals the intensity dependence of fluorescence and, hence, the number of excitation photons required to generate emission. Optimal laser intensities for detection were (32) In studies that are not limited to the small sample sizes analyzed in this work, concentration detection limits for two-photon fluorescence analysis could be improved substantially using a variety of approaches. In one strategy, the submicrometer laser focal spot position could scanned with galvinometer-driven mirrors to probe the outlet of much larger inner diameter capillaries. Alternatively, the size of the laser spot could be increased by underfilling the back aperture of the high NA objective (producing a Gaussian focus). In this approach, the maximum size of the probe area (
