Indirect Fluorescence Detection of Amino Acids on Electrophoretic

CE separations were performed using a 37 cm × 50 μm or 67 cm × 50 μm ... and fluorescence detection has been described previously.3 Safety note: P...
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Anal. Chem. 2000, 72, 2765-2773

Indirect Fluorescence Detection of Amino Acids on Electrophoretic Microchips Nicole J. Munro,† Zhili Huang,† David N. Finegold,‡ and James P. Landers*,§,|

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, Division of Pediatric Endocrinology, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213, Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, and Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22901

Microfabricated devices enable rapid separations of a variety of clinically significant analytes, including DNA, proteins, and amino acids. However, absorbance detection has been difficult to achieve on these devices, prohibiting analysis of nonfluorophore-bearing or nonfluorescently tagged analytes. An alternative detection technique exploiting indirect fluorescence has been adapted to the electrophoretic microchip to provide fast analysis of amino acids, bypassing the need for absorbance detection or fluorescence derivitization procedures. Nineteen of the standard amino acids could be detected with an average detection limit of 32.9 µM (∼1.6 amol). Despite the fact that the detection sensitivity was lower than that achievable by labeling the amino acids with fluorescein isothiocyanate (∼1 nM), circumventing sample preparation and the difficulties inherent with tagging complex samples make this technique attractive for a variety of assays where sensitivity is not critical. To demonstrate the applicability to real sample matrixes, the analysis of urine with elevated amino acid levels is used as a model system where the elevated levels are indicative of a variety of pathologies including amino acid metabolism disorders and kidney malfunction. The minimal sample handling and rapid separations achievable by employing indirect detection on microchips provides the potential for highthroughput applications for certain amino acid analyses. Electrophoretic microchips have proven to be an efficient embodiment of their parent technique, capillary electrophoresis (CE). Potential clinical applications have been described,1 and separations of serum proteins2 and PCR products3-5 have been * Corresponding author: (phone) 804-243-8658; (fax) 804-243-8852 (e-mail) [email protected]. † University of Pittsburgh. ‡ Children’s Hospital of Pittsburgh. § University of Virginia. | University of Virginia Health Sciences Center. (1) Colyer, C. L.; Tang, T.; Chiem, N.; Harrison, D. J. Electrophoresis 1997, 18, 1733-41. (2) Colyer, C. L.; Mangru, S. D.; Harrison, D. J. J. Chromatogr., A 1997, 781, 271-76. (3) Munro, N. M.; Snow, K.; Kant, J.; Landers, J. P. Clin. Chem. 1999, 45, 1906-17. (4) Chen, Y.-H.; Wang, W.-C.; Young, K.-C.; Chang T.-T.; Chen, S.-H. Clin. Chem. 1999, 45, 1938-43. 10.1021/ac9914871 CCC: $19.00 Published on Web 05/18/2000

© 2000 American Chemical Society

demonstrated. On-chip detection has relied predominantly on fluorescence, which is easily attainable for DNA fragments through fluorescent intercalators or fluorescently labeled primers in the case of PCR products. However, fluorescence detection of most other clinically significant analytes is not as easily facilitated. While proteins, hormones, and other (small) druglike molecules can be detected via UV absorbance using conventional capillary systems, this detection mode has been difficult to integrate into the microchip format.6 Channel depths remain shallow using current microfabrication techniques, minimizing potential absorbance path lengths. In addition, a number of clinically significant analytes, including most amino acids and simple ions, have no convenient chromophore and are, therefore, difficult to probe using molecular spectroscopy techniques. Fluorescence detection is recognized to be one of the most sensitive detection schemes for microminiaturized electrophoresis.7 However, to detect many nonfluorophore bearing analytes, derivitization of the analytes of interest with an appropriate fluorophore must be accomplished. Unfortunately, established chemistries for derivitization often lack specificity in the presence of complex mixtures, producing heterogeneous labeling.8,9 These problems appear to be exacerbated when an attempt is made to label analytes present at low concentrations. Three modes of fluorescence derivitization have been employed for detection in capillary electrophoresis, precolumn, postcolumn, and on-column, and all have been discussed thoroughly in the literature (for example refs 10 and 11).10,11 Precolumn derivitization has been used most extensively since reaction times, conditions, and cleanup (if necessary) are unrestricted. However, this has not easily been integrated into a rapid, automated analysis. Postcolumn derivitization has not been as utilized extensively due to restricted reaction conditions and rates and reduction of volume/length considerations when the technique is transferred from HPLC (5) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-86. (6) Liang, Z. H.; Chiem, N.; Ocvirk, G.; Tang, T.; Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 1040-46. (7) Pentoney, S. L.; Sweedler, JV. In Handbook of Capillary Electrophoresis; Landers, J. P. Ed.; CRC Press: Boca Raton, FL, 1994; pp 379-423. (8) Krull, I. S.; Strong, R.; Sosic, Z.; Cho, B. Y.; Beale, S. C.; Wang, C. C.; Cohen, S. J. Chromatogr., B 1997, 699, 173-208. (9) MacTaylor, C. E.; Ewing, A. G. Electrophoresis 1997, 1812-13, 2279-2290. (10) Bardelmeijer, H. A.; Lingeman, H.; de Ruiter, C.; Underberg, W. J. M. J. Chromatogr., A 1998, 807, 3-26. (11) Smith J. T. Electrophroresis 1997, 18, 2377-2392.

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where it has been used successfully. The most promising derivitization technique for rapid, automated amino acid analysis via CE is on-column derivitization where the analyte is introduced into the capillary between two plugs of labeling reagent. Despite restrictions with the reaction time and conditions, this method has been successfully applied to several amino acids analyses.10 Each of the labeling modes possesses specific advantages and disadvantages that must be weighed for each application to obtain the optimal assay. When fluorescence derivitization for microchip-based analysis is involved, a clear advantage is obtained over CE due to the ability to integrate reaction chambers and, if needed, heaters onto the microchip. This reduction in sample handling and human error could potentially increase the reproducibility of the assay, in addition to reducing analysis time. However, limitations still exist in the availability of fluorogenic reagents that provide rapid, quantitative reactions while yielding products that are stable for analysis without interfering byproducts.10,11 This is evidenced in the literature by the wide variety of reagents used, with each fulfilling the specific assay criteria while working within the limitations of the reagent.10 In addition, separation of the amino acids is usually exasperated by the addition of an identical bulky moiety (in comparison to most amino acids), reducing analyte differences.10,11 Nevertheless, separation of derivatized amino acids on microchips employing on-chip, precolumn derivitization12 and postcolumn reaction13 has been achieved. As an alternative detection technique, Hjerte´n et al.14 employed a more universal mode of detection for the electrophoretic analysis of inorganic and organic ions. This method involves indirect detection and utilizes a buffer containing a chromophore/fluorophore that functions as a probe. Due to local charge neutrality requirements, analytes of like charge displace the probe, lowering the local concentration of probe in the analyte band and, therefore, lowering the observed signal. This detection method has been adapted to CE and employed for a variety of clinically relevant analytes including inorganic anions,15 amino acids,16-19 lipids,20 and sugars.21 While a UV-absorbing probe is traditionally used, fluorescent probes have also been employed.22-24 Indirect fluorescence detection is ideal for microchip applications due to the limitations associated with absorbance detection and fluorescent tagging as discussed above. In this paper, we discuss the application of indirect fluorescence detection to the electrophoretic microchip format. We show that indirect fluorescence detection can be translated from the capillary platform to the microchip format (12) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W.; Ramesy, J. M. Anal. Chem. 1994, 66, 4127-4132. (13) Jacobson, S. C.; Koutny L. B.; Hergenroder R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (14) Hjerte´n, S.; Elenbring, K.; Kilar, F.; Liao, J.-I.; Chen, A. J. C.; Siebert, C. J.; Zhu, M.-D. J. Chromatogr. 1987, 403, 47-61. (15) Jones, W. R. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994; pp 162-167. (16) Lee, Y.-H.; Lin, T.-I. J. Chromatogr., A 1994, 680, 287-297. (17) Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 60, 1832-34. (18) Lee, Y.; Lin, T. J. Chromatogr., A 1995, 716, 335-46. (19) Chen, H.; Xu, Y.; Ip, M. P. C. J. Liq. Chromatogr. 1997, 20, 2475-93. (20) Wang, T.; Wei, J.; Li, S. F. Electrophoresis 1998, 19, 2187-92. (21) Richmond, M. D.; Yeung, E. S. Anal. Biochem. 1993, 210, 245-48. (22) Chao, Y.; Whang, C. J. Chromatogr., A 1994, 663, 229-37. (23) Xue, Q.; Yeung, E. S. J. Chromatogr., A 1994, 661, 287-295. (24) Huang, Y. M.; Whang, C. W. Electrophoresis 1998, 19, 2140-44.

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where high-throughput capabilities exist. In addition, we demonstrate the use of this indirect detection methodology for the fast analysis of amino acids in urine. Abnormal amino acid concentrations in urine are indicative of several pathologies, such as amino acid metabolism disorders and kidney malfunction.25 The former, typically due to inborn errors of metabolism, can result in catastrophic developmental problems in newborns, resulting in mental retardation and reduced life expectancy.26 Rapid diagnosis of these diseases is essential to control the extent of bodily damage; however, newborn screening is currently not performed. The described methodology highlights the potential for highthroughput, rapid analysis (120 s) of minimally processed urine samples for routine diagnostic screening. MATERIALS AND METHODS Materials. Sodium carbonate was obtained from EM Science (Gibbstown, NJ). Hexadecyltrimethylammonium bromide (CTAB) and L-amino acid standards were obtained from Sigma Chemicals (St. Louis, MO), in addition to the following urine component standards: 1-methyl-L-histidine, 3-methyl-L-histidine, β-alanine (βala), L-ornithine, DL-β-aminoisobutyric acid, R-aminoadipic acid, O-phospho-L-serine, L-R-amino-n-butyric acid, taurine (tau), Lhomocarnosine, O-phosphorylethanolamine, and ethanolamine. Hexadecyltrimethylammonium hydroxide (CTAOH) was purchased from Aldrich Chemicals Co. (Milwaukee, WI). The disodium salt of fluorescein was obtained from Acros Chemicals (Fair Lawn, NJ). Polyimide-coated capillaries (50 µm i.d. × 360 µm o.d.) were purchased from Polymicro Technologies (Phoenix, AZ). Separation buffers were made from stock solutions of fluorescein, sodium carbonate, and, if indicated, CTAOH. The pH was adjusted with 0.1 M HCl or 0.1 M NaOH to the indicated values. All buffers were made with purified water (Millipore, Bedford, MA) and filtered through a 0.2-µm filter before use. Over several hours, the pH of the run buffer solution would decrease due to CO2 absorption; therefore, the run buffer was made fresh from stock solutions prior to use. Individual stock solutions of the amino acids were prepared in water, and if necessary, HCl or NaOH was added until the compound dissolved. The standard amino acid sample for electrophoretic analysis consisted of the 20 standard amino acids at individual concentrations of 0.4 mM prepared in 0.5 mM fluorescein in water before analysis. Fluorescein isothiocyanate (FITC)tagged amino acids were prepared as previously described.27 Urine samples were filtered (0.2 µm, Millipore) and then diluted 1:5 for analysis in a 67-cm capillary or 1:10 for analysis in a 37-cm capillary. A 0.5 mM concentration of fluorescein was maintained in all urine samples. For microchip analysis, the filtered samples were diluted 1:10 with a final concentration of 1 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH. When not in use, all standards were stored at 4 °C and all urine specimens were stored at -18 °C. HPLC analysis of urine samples was performed using a previously described methodology.28 (25) Ross D. L.; Neely A. E. Textbook of Urinalysis and Body Fluids; AppletonCentury-Crofts: Norwalk, CN, 1983. (26) Nyhan, W. L. Abnormalities in amino acid metabolism in clinical medicine; Appleton-Century-Crofts: Norwalk, CN, 1984. (27) Jin, L. J.; Rodriguez, I.; Li, S. F. Y. Electrophoresis 1999, 20 (7), 15381545.

Capillary Electrophoresis Instrumentation. A Beckman P/ACE System 2210 (Beckman Instruments, Fullerton, CA) was used for CE analysis. Fluorescence detection employed a P/ACE System Laser Module 488 with a P/ACE LIF detector that excites at 488 nm and collects emission at 520 ( 10 nm. A laser power of 10.5 µW was incident on the capillary. Instrument control and data collection were performed with an IBM 486 computer utilizing System Gold software (V. 8.1). Electrophoresis was performed in the indirect detection mode that inverts the fluorescence signal. CE-LIF Analysis. CE separations were performed using a 37 cm × 50 µm or 67 cm × 50 µm capillary (effective lengths of 30 and 60 cm, respectively). Capillaries were initially rinsed with 20 column volumes of water, 20 column volumes of 0.1 M NaOH, and again 20 column volumes of water. Separations involved the following method: 2-min prerinse with nonelectrophoresed buffer, 1-s pressure injection of sample followed by a 2-s pressure injection of run buffer. Separation followed at 224 V/cm using normal polarity (inlet anode) or reversed polarity (inlet cathode) if CTAOH was employed. Capillary temperature was maintained at 20 °C. The capillary was rinsed with 5 column volumes of water and 2 column volumes of 0.1 M NaOH between runs. Peaks were identified by spiking the samples with amino acid standards. Standard equations were used to calculate injection volumes.29 Peak analysis was performed using Grams/386 software. Electrophoretic Microchip Instrumentation. Instrumentation used for microchip voltage control and fluorescence detection has been described previously.3 Safety note: Proper safety precautions should be taken to isolate the high voltage to avoid contact. Direct eye exposure to laser radiation will damage eye tissue; therefore, a contained system should be used. Electrophoretic chips were fabricated by the Alberta Microelectronics Center (Edmonton, AB, Canada) and employed a simple crosschannel layout as described previously.30 Amino Acid Analysis via Microchip Electrophoresis. Microchip sample injection was performed by applying a -400 V (333 V/cm) potential across the sample and sample waste reservoirs, with the sample at ground for normal polarity runs for 15 s. Subsequent separation involved placing the sample and sample waste to ground and applying 300 V to the inlet and -1200 V to the outlet (183 V/cm). For runs employing CTAOH and reversed polarity, optimal sample injection required grounding the sample and applying 500 V (417 V/cm) to the sample waste for 15 s while separation required placing the sample and sample waste to ground, the inlet to -300 V, and the outlet to 1200 V. An effective separation length of 5.5 cm was used, and fluorescence was collected at 10 Hz. Grams/386 was used to invert, filter, and analyze the electropherograms. Injection volumes were calculated from separation and injection channel intersection geometry. RESULTS AND DISCUSSION A. Optimization of Capillary- and Microchip-Based Electrophoresis of Amino Acids. Indirect absorption detection for amino acid analysis via CE has been achieved using a variety of (28) Drescher, M. J.; Medina, J. E.; Drescher, D. G. Anal. Biochem. 1981, 116 (2), 280-286. (29) Landers, J. P., Ed. Handbook of Capillary Electrophoresis; CRC Press: Boca Raton, FL, 1994; pp 868-869. (30) Hofga¨rtner, W. T.; Hu ¨ hmer, A. F. R.; Landers, J. P.; Kant, J. A. Clin. Chem. 1999, 45, 2120-2128.

UV chromophores including salicylate,19 benzoate,19 and 4-(N,N′dimethylamino)benzoic acid (DMAB) and p-aminosalicylic acid.16,18 Lee and Lin18 demonstrated the highest resolution CE separation/ indirect absorption detection of amino acids to date by closely matching the electrophoretic mobility of the probe to as many amino acids as possible. For optimal separation, they employed DMAB as the probe to identify 19 amino acids, with separation of all but isoleucine and leucine in 55 min. Prior literature describing the CE separation and detection of amino acids via indirect fluorescence detection is sparse, with the only report being that by Kuhr and Yeung,17 who separated several amino acids using salicylate as the background electrolyte. This results, at least in part, from the fact that few fluorophores have been investigated for their effectiveness for indirect detection in CE due to solubility, charge, and pH fluorescence dependencies, as well as compatibility requirements with existing fluorescence systems. Fluorescein has been utilized extensively as a common fluorophore as a result of the solubility of the disodium salt in aqueous buffers and the fact that it can be excited with an affordable, readily available argon ion laser. Additional features that make it attractive include its low cost and negative charge over a wide pH range (pH >7).31 As a result of these attributes, fluorescein was evaluated as a fluorophore for indirect amino acid detection. The amine groups on the standard amino acids range in pKa from 6.13 to 10.96 with an average pKa of 9.22.32 Above this pKa, most amino acids have a negative charge (with the exception of lysine and arginine due to positively charged R groups) and, therefore, can be detected using indirect detection with a negatively charged probe. To achieve adequate separation, compromise charge to mass differences must be reached between the individual pKa values and total deprotonation conditions. Consequently, a separation buffer with relatively high pH is required. Buffer choices are limited in this pH range; however, carbonate has been shown to be an effective buffer for amino acid analysis17,19 and was used here. To achieve optimal separation of amino acids in conjunction with indirect fluorescence detection, the buffer concentration must be minimized but still provide adequate buffering capacity. High-concentration buffers reduce sensitivity by competing with the probe to displace the analyte molecules, thus reducing signal differences between the background and analyte bands.15 Consequently, compromises must be made between resolution and sensitivity when buffer concentration is being optimized. Using a two-component buffer system comprising carbonate (buffer) and fluorescein (probe), a concentration range of 0.25-1.5 mM was tested for each component. Taking both resolution and buffering capacity into consideration, the results converged at 1 mM carbonate and 0.5 mM fluorescein as the optimal combination for amino acid analysis. Since this concentration of fluorescein far exceeded that necessary for effective detection (detector limit was reached), the laser power was reduced to bring the signal intensity into the detectable range of the capillary electrophoresis system. Optimization of the separation buffer pH was also performed over a range of 8.811.2. Figure 1A provides a characteristic separation of nine amino (31) Sjoback, R..; Nygren, J.; Kubista, M. Spectrochim. Acta A 1995, 51A (6), L7-L27. (32) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993; p 113.

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Figure 1. Indirect detection of select amino acids for capillary and microchip electrophoresis: (A) capillary and (B) microchip electropherograms for nine amino acids. Buffer: 1.0 mM sodium carbonate and 0.5 mM fluorescein at pH 11.0. Capillary conditions: leff 30 cm, 1-s sample injection, and 224 V/cm separation voltage. Microchip conditions: leff 5.5 cm, 15-s injection at 333 V/cm, and 183 V/cm separation voltage. Amino acid concentrations: 0.4 mM in 0.5 mM fluorescein. Abbreviations: K, lysine; P, proline; L, leucine; H, histidine; T, threonine; A, alanine; S, serine; G, glycine; Y, tyrosine.

acids in a capillary with an effective length (Leff) of 30 cm using 1.0 mM sodium carbonate and 0.5 mM fluorescein buffer at pH 11.0. This pH was found to be optimal for the majority of the standard amino acids, excluding the diacidic amino acids (cysteine, glutamic acid, aspartic acid), which migrated as two poorly resolved peaks (data not shown). These optimized conditions have been translated to the microchip to demonstrate the utility of indirect fluorescence detection for microchip analysis of nonfluorophore-bearing analytes. Comparable resolution was observed on the microchip (Figure 1B), despite the inherently shorter separation distance (5.5 cm). Reoptimization of the separation conditions for the microchip from that determined optimal on the capillary was deemed unnecessary due to the similar resolution obtained. As stated earlier, the diacidic amino acids (cysteine, glutamic acid, aspartic acid) were not adequately resolved under these conditions. However, lowering the pH to 9.0 allowed for separation of these amino acids without interference from other species both in a capillary and on the microchip (Figure 2). 2768 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

Figure 2. Analysis of diacidic amino acids, glutamic acid, aspartic acid, and cysteine: (A) capillary and (B) microchip electropherograms. Buffer: 1.0 mM sodium carbonate and 0.5 mM fluorescein at pH 9.0. Amino acid concentrations: 0.4 mM in 0.5 mM fluorescein. Abbreviations: C, cysteine; E, glutamic acid; D, aspartic acid. Other conditions as in Figure 1.

As with many separations employing indirect detection, optimal resolution of all the amino acids involved the use of a dynamically coated capillary/microchannel wall and a polarity that employs the inlet as the cathode and the outlet as the anode (i.e., reversed polarity). Dynamic coatings that reverse EOF are used to provide more rapid separations through a co-EOF condition (the apparent electrophoretic mobility of the analytes and the EOF are in the same direction).33,34 Cetyltrimethylammonium bromide (often referred to as hexadecyltrimethylammonium bromide) is employed commonly as the dynamic coating and was evaluated here for the amino acid separation. However, higher resolution was obtained with the use of the hydroxylated version, CTAOH, which is alternatively used to eliminate the bromide, which can function as an additional anion to compete with the probe.33 Studies optimizing the CTAOH concentration showed that reproducible separations could be obtained at concentrations ranging from 0.2 to 0.4 mM, but that the highest resolution was obtained at the lower end of this range (0.2 mM). Using these CTAOH concentra(33) Harakuwe, A. H.; Haddad, P. R. J. Chromatogr., A 1999, 834, 213-232. (34) Doble, P.; Andersson, P.; Haddad, P. R. J. Chromatogr., A 1997, 770, 291300.

Figure 3. Separations of select amino acids using CTAOH as a dynamic coating: (A) capillary and (B) microchip electropherograms of eight amino acids employing 0.2 mM CTAOH in the running buffer with 1.0 mM sodium carbonate and 0.5 mM fluorescein at pH 10.3. Capillary conditions: leff 30 cm, 1-s sample injection, 224 V/cm (reversed polarity) separation voltage, and sample amino acid concentrations of 0.4 mM in 0.5 mM fluorescein. Microchip conditions: leff 5.5 cm, 15-s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, and sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Abbreviations: Y, tyrosine; G, glycine; S, serine; T, threonine; A, alanine; H, histidine; F, phenylalanine; L, leucine.

tion restrictions, a pH of 10.3 was found to be optimal for the amino acid separation employing a 1.0 mM carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH buffer. These separation conditions are similar to those reported by Chen et al.,19 where 5 mM carbonate, 2.0 mM salicylate (the probe), and 0.15 mM myristyltrimethylammonium bromide (MTAB; a dynamic coating) at pH 10.7 were used. Figure 3 illustrates the separation obtained with a standard mixture containing eight amino acids when CTAOH is employed. Microchip electrophoresis also yielded effective resolution of the representative amino acids, although there was a decrease in the signal-to-noise ratio (S/N) when compared to the capillary. This decrease in sensitivity is due, in part, to the reduced volume of sample injected into the microchannel (50 pL) when compared to that injected into the capillary (1.43 nL). Separation temperature has also been shown to effect noise levels when indirect detection is employed35 and may contribute to the

decreased S/N for the microchip separation since it was performed at ambient temperature (∼24 °C) whereas the capillary experiments were performed at 20 °C. As a result of this reduction in S/N, several parameters of the detection system were evaluated for optimization. Optimization of the detection system for signal strength included varying the laser power and, as needed, the PMT gain so the background signal was within range. No significant trends could be identified until laser power was reduced to an estimated 0.064 µW (at the channel) where S/N decreased; therefore, 6.4 µW was used for data collection. Detection sensitivity was also tested using different diameter pinholes (400, 200, 100, 50 µm) to vary the confocality of the system. As a result of the high concentration of fluorescein needed to function as the background electrolyte, “diluting” of the analyte band signal may occur if the size of the detection spot is not sufficiently limited.15 However, no improvement was found when the detection volume was restricted through the use of smaller pinholes; therefore, the 200-µm pinhole was used for routine data collection. Beyond detection system optimization, the microchannel structure could also be modified to potentially improve S/N; increasing the injection channel size would allow for an increased injection volume and, therefore, increased S/N. However, the analysis in its present form allows for efficient separation of amino acids standards with detection limits amenable to many applications. B. Capillary and Microchip-Based Electrophoresis of 20 Amino Acids. The optimized conditions used to yield amino acid separations shown in Figure 3 were employed for the separation and detection of 20 amino acids in a standard mixture. Extending the capillary Leff from 30 to 60 cm allowed for 19 of the 20 amino acids to be identified (Figure 4A). Arginine could not be identified due to insufficient deprotonation at the separation pH (R group amine pKa ) 10.76). Most of the 19 amino acids were, at least, partially resolved with the exceptions being leucine and isoleucine, methionine and glutamine, and phenylalanine and valine. The comigration of leucine and isoleucine was expected due to their structural similarities. This is exemplified by Lee and Lin,18 who were unable to resolve these two amino acids even when employing modified cyclodextrins in the separation buffer. The comigration of the remaining amino acids (methionine and glutamine, phenylalanine and valine) may be the result of a poor mobility match with the probe (fluorescein). The mobility of fluorescein is well matched to that of the amino acids eluting near glycine, as demonstrated by the narrow peak widths. However, the match with the probe is less optimal for those amino acids with lower mobility, such as tryptophan, as evidenced by the asymmetric appearance of these peaks. Unfortunately, the chemical diversity of amino acids, specifically their wide range of mobilities, makes it very unlikely that any one probe will be optimal for all 20 amino acids. This is only exacerbated by the fact that the probe must also fulfill the practical requirements discussed earlier. It is noteworthy that the diacidic amino acids (as shown in Figure 4) display inverted peaks (which are actually positive peaks since the electropherograms have been inverted). This phenomenon has been observed by several groups with a variety of analytes,19,22,36 and several explanations have been offered, which (35) Xu, X.; Kok, W. T.; Poppe, H. J. Chromatogr., A 1997, 786, 333-345.

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Figure 4. Indirect detection of the standard amino acids for capillary and microchip electrophoresis: (A) 67-cm capillary, (B) 37-cm capillary, and (C) microchip electropherograms for 19 amino acids. Buffer: 1.0 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH at pH 10.3. Capillary conditions: leff 30 or 60 cm, 1-s sample injection, 224 V/cm (reversed polarity) separation voltage, and sample amino acid concentrations of 0.4 mM in 0.5 mM fluorescein. Microchip conditions: leff 5.5 cm, 15-s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, and sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Abbreviations: D, aspartic acid; E, glutamic acid; C, cysteine; tau, taurine; Y, tyrosine; G, glycine; S, serine; N, asparagine; T, threonine; A, alanine; M, methionine; Q, glutamine; H, histidine; F, phenylalanine; V, valine; L, leucine; I, isoleucine; W, tryptophan; P, proline; K, lysine.

were tested for the separation system employed here. Inverted peaks have been attributed to lessened self-quenching in the analyte band;37 however, for the system employed here, 0.5 mM fluorescein was found to be in a concentration range with a linear detector response (data not shown). Shamsi et al.36 attributed their inverted peaks to the localized decreases in pH associated with anion analyte bands and the pH dependency of their fluorophore, flavin mononucleotide. The pH dependency of fluorescein in the presence of CTAOH was, therefore, tested; however, the expected decrease in fluorescence intensity was observed with decreasing pH, indicating fluorescence intensity in lower pH bands should be decreased, not increased as would be necessary to invert the peaks (data not shown). Consequently, mechanisms proposed to explain inverted peaks are not applicable to this separation system; nonetheless, separation and identification of the three diacidic amino acid inverted bands was achieved. (36) Shamsi, S. A.; Danielson, N. D.; Warner, I. M. J. Chromatogr., A 1999, 835, 159-68. (37) Jurkiewicz, K.; Dasgupta, P. K. Anal. Chem. 1987, 59, 1362.

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Procedures used for CE separation of anions typically employ long capillaries (>60 cm) to allow for optimal resolution of the analytes.15 With the goal of translating these conditions to the microchip, the 67-cm capillary (Leff ) 60 cm) (Figure 4A) was reduced to 37 cm (Leff ) 30 cm) (Figure 4B), which, under the same conditions, yielded a resolution that was slightly reduceds this is indicated by comigration of alanine with methionine and glutamine. However, the resolution of the remaining amino acids was retained and a significant reduction in analysis time is achieved. When the effective separation length is reduced even further by electrophoresing on the microchip in a channel that had a Leff ) 5.5 cm (Figure 4C), the resolution was found to correlate well with that obtained with the 30-cm effective length capillary (Figure 4B). There was a slight reduction in resolution, as evidenced by the comigration of histidine with phenylalanine and valine, and only partial resolution of glycine and serine. However, partial resolution of alanine, methionine, and glutamine was observed. As indicated by the capillary experiments, the resolution could be improved by increasing the effective length via the use of serpentine structures. Although, even in the absence of such design changes, the resolution demonstrated here presents opportunities for a variety of applications. Detection limits are an important consideration when the components of biological matrixes are analyzed, particularly when they are present at low or trace concentrations. Due to the displacement phenomenon inherent to indirect detection, the detection mode is typically less sensitive than direct fluorescence detection. CE concentration limits of detection for amino acids under the conditions yielding the separation in Figure 3A range from 2.3 to 47.8 µM, with an overall average of ∼12.0 µM (S/N ) 3) for the particular amino acids evaluated here. The average detection limit remained relatively constant when all 20 amino acids were considered (12.3 µM). The detection limits were higher with the microchip separations, ranging from 9.35 to 67.7 µM with an average of 32.9 µMsthis translates to a detection limit of ∼1.6 amol. Detection limits were also evaluated for conditions not involving the addition of CTAOH to the separation buffer (see Figure 1). It was observed that the traditional yellow color typical of concentrated fluorescein solutions changed to a yellow-orange hue in the presence of CTAOH. Biswas et al.38 showed the emission maximum of fluorescein after the addition of CTAB undergoes a red shift from 513 to 523 nm. However, this did not largely affect the detection limits, which showed only a modest improvement (24% increase) under conditions not employing CTAOH. The average microchip concentration limit of detection for the amino acids evaluated here (32.9 µM) was well within the range for many amino acid concentrations expected in biological fluids such as urine and serum.39 However, there is no question that much lower detection limits can be obtained by fluorescent tagging of the amino acids. Using 1 mM carbonate as the buffer with the other conditions as in Figure 1B, concentration limits of detection for FITC-alanine and FITC-isoleucine on the microchip were determined to be 0.58 and 0.92 nM, respectively. This (38) Biswas, S.; Bhattacharya, S. C.; Sen, P. K.; Moulik, S. P. J. Photochem. Photobiol. A: Chem. 1999, 123, 121-128. (39) Shapira, E.; Blitzer, M. G.; Miller, J. B.; Africk, D. K. Biochemical Genetics: A Laboratory Manual; Oxford University Press: New York, 1989; pp 9697.

represents a differential of over 5 orders of magnitude between the indirect and direct fluorescence detection approaches. This is comparable to the studies of von Heeren et al.,40 who achieved detection limits of 3.3 nM for FITC-Arg. High-resolution electrophoresis of FITC-tagged amino acids have also been achieved in a capillary by both Nouadje et al.41 and Takizawa and Nakamura,42 where 17 and 19 amino acids, respectively, were resolved. On the microchip, von Heeren et al.40 separated six amino acids on a cyclic microstructrure while Hutt et al.43 showed the separation of several chiral pairs of FITC-labeled amino acids. In the former work, FITC labeling of urinary components was carried out to tag the amino acids, but no attempt was made to identify the labeled peaks. However, the potential of separating derivatized urinary amino acids in the capillary can be seen in the work by Zhu and Kok,44 where postcolumn derivitization with o-phthalaldehyde (OPA) allowed separation and identification of seven amino acids in normal urine. While the resolving power of the direct and indirect detection approaches appear similar, one must trade off detection sensitivity in indirect detection with the time and effort required by the labeling process or the complicated labeling instrumentation which must be engineered for direct detection. On the other hand, an indirect approach to detection lends itself to rapid, lowcost sample analysis with simplified profiles that may offer an alternative method for the detection of many analytes. C. Detection of Amino Acids in Urine. The amino acid content of urine is an important indicator of amino acid balance in the body. Elevation in the concentration of certain amino acids in the urine can be indicative of an abnormality in amino acid metabolism, renal transport, or diet of the individual. Of particular importance is the identification of elevated amino acid levels as a result of inborn errors in amino acid metabolism. These errors usually result in nonfunctional enzymes in a manner that eventually affects amino acid catabolic pathways. The inability to process substrate through the next metabolic step results in increased levels of the affected amino acid(s) or their metabolites in the serum. This increased concentration is eventually reflected in the urine once the renal threshold for these compounds is reached. While these inborn errors in metabolism can lead to mental retardation if not detected early, the disease can be managed with proper dietary changes and compliance monitoring. Currently, neonatal amino acid screening is not performed routinely and analysis is only performed after symptomatic indicators are observed (lethargy, poor feeding, odors). The traditional methodology involves HPLC analysis for the interrogation of 40 amino acids potentially found in urine.28 Microchip separation of amino acids introduces the possibility of a more rapid and cost-effective amino acid analysis that would be amenable to high-throughput screening. Normal urine contains amino acids at concentrations ranging from 0 to 24 mmol/Lsthis includes the standard 20 amino acids as well as many metabolites.39 The analysis profile of a normal urine specimen in a capillary with a Leff ) 60 cm is shown in Figure (40) Von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (41) Nouadje, G.; Rubie, H.; Chatelut, E.; Canal, P.; Nertz, M.; Puig, P.; Couderc, F. J. Chromaogr., A 1995, 717, 293-298. (42) Takizawa, K.; Nakamura, H. Anal. Sci. 1998, 14, 925-928. (43) Hutt, L. D.; Glavin, D. P.; Bada, J. L.; Mathies, R. A. Anal. Chem. 1999, 71, 4000-4006. (44) Zhu, R.; Kok, W. T. J. Chromatogr., A 1995, 716, 123-133.

Figure 5. Analysis of normal urine specimen via capillary electrophoresis (67-cm capillary). Peak assignments represent coellution with amino acid standards. Labels represented by arrows indicate where the amino acids would migrate if present in the urine specimen. Sample diluted 1:5 in water. Conditions and abbreviations as in Figure 4.

5. No sample preparation was performed beyond filtering the urine and diluting it 1:5 in water. The dilution was needed due to the ∼150 mM NaCl present in urine, in addition to lower concentrations of other inorganic ions (potassium, bicarbonate, etc.). This ionic strength varies dramatically in comparison with that of the run buffer; therefore, dilution was required to achieve the desired resolution. Peak assignments were accomplished by individually spiking the diluted urine with each of the 20 standard amino acids as well as several other metabolites described in the Materials and Methods section. Not all peaks could be identified. The assignments were made based on where the spiked standard migrated (e.g., taurine, tyrosine, glycine, etc.) or where the amino acids would migrate if a peak is absent in the urine (e.g., phenylalanine, valine, β-alanine). When a 37-cm capillary was employed, resolution of the urinary components was lowered slightly (Figure 6A) in comparison with that observed in Figure 5. The same injection time (1 s hydrodynamic) was utilized with both the 67- and 37-cm capillaries. However, due to lower back pressure, a much larger volume was injected into the 37-cm capillary (1.43 nL) than the 67-cm capillary (0.79 nL). Since the injection time could not be reduced below 1 s, urine specimens required 1:10 dilution for analysis in the 37cm capillary to compensate for this larger injection volume and to maintain optimal resolution and signal-to-noise ratio. Microchip analysis of a normal urine sample yielded a profile similar to that observed with the 37-cm capillary; however, as discussed earlier, a significant reduction in S/N was observed (Figure 6D). Nonetheless, several peaks could be identified with putative assignments for glycine, serine, threonine, alanine, methionine, glutamine, isoleucine, and leucine possible. It is noteworthy that the resolution (as observed in Figure 4) also decreased as the capillary was shortened from 67 to 37 cm and then to 5.5 cm on the microchip displaying again the potential for higher resolution on the microchip by lengthening the channel. Analytical Chemistry, Vol. 72, No. 13, July 1, 2000

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Figure 6. Separation of normal and abnormal urine samples via capillary (37 cm) and microchip electrophoresis. Capillary electrophoresis of (A) the normal specimen analyzed in Figure 5, (B) abnormal urine sample 1, and (C) abnormal urine sample 2. Microchip separation of (D) the specimen analyzed in Figure 5, (E) abnormal urine sample 1, and (F) abnormal urine sample 2. Conditions and abbreviations as in Figure 4, except urine samples were diluted 1:10 in water with 0.5 mM fluorescein. For microchip analysis, the final concentration also included 1 mM sodium carbonate and 0.2 mM CTAOH.

Injection methods on microchips vary from that on the capillary.45,46 Use of reduced/reversed EOF through channel coatings requires a different injection method when compared to the hydrodynamic injection employed for these studies on the capillary. EOF measured for the system employed in Figure 1 was 5 times larger than the reversed EOF measured under the conditions with CTAOH, as in Figure 3; however, when longer injections were used to compensate for the lowered EOF, resolution decreased. Concomitantly, sample pH was varied, ensuring the amino acids were above their isoelectric point so the injection would be optimal. Dilution of the urine with 10 mM sodium carbonate at pH 12.0 and the addition of CTAOH to a concentration of 0.2 mM were necessary for consistent replicate analysis. Although dilution of the urine prior to analysis reduces the signal-to-noise ratio, the detection of amino acids in abnormal urine specimens was feasible. Two urine specimens, verified as abnormal by HPLC-based amino acid analysis, were analyzed electrophoretically in a capillary (Figure 6B,C) and on the microchip (Figure 6E,F)sspiking experiments allowed for the identification of several amino acids. The first sample (Figure 6B,E) was from a patient suspected of having nephropathic cystinosis with the onset of Fanconi syndrome. Among other things, this results in a generalized hyperaminoaciduria (elevated concentrations of amino acids in urine). HPLC analysis indicated elevations of a number of amino acids including alanine, valine, glutamine, and isoleucine. The second sample was from a patient exhibiting symptomatic (45) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (46) Fan Z. H.; Harrison D. J. Anal. Chem. 1994, 66, 177-184.

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indicators for amino acid metabolic disorders, but HPLC analysis indicated only mild elevation of several amino acids. Through capillary and microchip electrophoretic analysis (Figure 6C,F), several amino acids (glycine, serine, threonine, histidine) could be identified that were determined to be elevated via HPLC analysis. It is unlikely that the method, as described here, would allow for the quantitative determination of amino acid levels, even in abnormal urine where concentrations can be elevated considerably. However, despite the lower sensitivity offered by indirect fluorescence detection, this method could allow for rapid detection of gross changes in urinary amino acid concentrations. One could envision, at some point in the future when commercial instrumentation was available, that a microchip-based system could function as a simple tool for the primary screening of a number of urinary amino acid abnormalities including metabolic diseases. CONCLUSIONS The ability to carry out indirect fluorescence detection on electrophoretic microchips not only enables the detection of nonchromophore-bearing analytes, but adds another mode to the detection arsenal of the analytical microchip platform. This detection method eliminates fluorescent labeling procedures and bypasses the difficulties experienced with absorbance detection on microchips. Using fluorescein as the background electrolyte and hexadecyltrimethylammonium hydroxide as a dynamic microchannel coating, 19 amino acids could be identified via microchip electrophoresis with an average detection limit of 32.9 µM (1.6 amol). Using elevated amino acids in urine as a model system, this methodology was extended to the microchip where

large abnormalities in urinary amino acid concentrations could be indicative of certain metabolic disease states. Although the detection limits for indirect detection are lower than those achievable with fluorescently labeled analytes, the lack of sample preparation and simplified electrophoretic profiles will make it an attractive alternative for many analyses.

for fluorescently tagging the amino acids for the detection limit study. We thank Dr. Jed Harrison (University of Alberta) for his early support in assisting us in understanding detection schemes for microchip electrophoresis. We also thank Beckman Coulter for CE instrumentation.

ACKNOWLEDGMENT We thank Lorna Cropcho (Children’s Hospital of Pittsburgh) for providing the abnormal urine HPLC analysis. We also thank Dr. Lianji Jin (Department of Chemistry, University of Virginia)

Received for review December 29, 1999. Accepted April 9, 2000. AC9914871

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