Characterization of Electrochemical Array Detection for Continuous

each spaced by 5 µm) and has been previously used in channel structures with internal heights as small as ∼21. µm. Here, the ability to differenti...
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Anal. Chem. 1997, 69, 3838-3845

Characterization of Electrochemical Array Detection for Continuous Channel Electrophoretic Separations in Micrometer and Submicrometer Channels Peter F. Gavin and Andrew G. Ewing*

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

Characterization of a microfabricated electrochemical array detection scheme used for continuous electrophoretic separations in narrow channels is described. The amperometric detector consists of 100 platinum microelectrodes (95 µm wide, 1.2-2 mm long, 0.12 µm high, each spaced by 5 µm) and has been previously used in channel structures with internal heights as small as ∼21 µm. Here, the ability to differentiate both mass and concentration changes of dopamine is demonstrated in 8-µm-internal height channel structures with electrochemical detection. Characterization of the array detector to provide insight into the nonuniform sensitivity observed with the microfabricated electrophoresis-electrochemical array detection technique is detailed. Approaches to circumvent the nonuniform sensitivity of the microelectrode array are described. Finally, we push the limits of the fabrication technology with the construction and use of submicrometer internal height (∼0.6 µm) rectangular channel structures. Novel developments and applications of capillary electrophoresis (CE) technology have forged its rapid growth as a highly efficient analytical separation technique in many areas of science.1-3 The low sample volume handling capabilities and highly sensitive detection schemes developed to date have made CE the separation technique of choice for qualitative and quantitative investigation of biological microenvironments.4-14 Several CE-based methods have been applied to the study of the contents of whole and subcellular compartments of cells with impressive results.5,10-13 However, most CE-based methods offer relatively limited temporal

resolution for dynamic biological environments, as injections are typically made in an incremental fashion. In our laboratories, an electrophoresis-based sampling and separation technique has been developed that can provide both the temporal resolution and chemical selectivity necessary for the investigation of constantly changing biological microenvironments.15-20 The fundamentals of the technique, termed channel electrophoresis, along with several useful applications, have recently been demonstrated.15-23 Briefly, a cylindrical electrophoresis capillary is used to continuously sample, but not separate, material from a sample environment and deliver it to a rectangular channel, where electrophoretic separation occurs. The sampling capillary is moved across the width of the channel entrance to provide continuous, time-resolved, low-volume (nanoliter to picoliter) sample introduction. The time resolution of the sampling process is preserved with the use of a spatially specific detector. Since migration inside the channel occurs in straight paths, the location of detection can be used to determine the position of the sampling capillary when the sample is introduced to the channel. Furthermore, the ability to determine both the time and duration of analyte contact with the entrance of the sampling capillary in channel electrophoresis has been demonstrated.18 One of the key components of the continuous sampling and separation technique is the detection scheme employed. Similar to detection methods in CE, sensitive, selective, and efficient detection schemes are essential, with the additional requirement of spatially resolved detection. Three approaches to site-specific detection have been demonstrated for channel electrophoretic analyses. Laser-induced fluorescence with excitation and emission fiber-optic arrays coupled to a linear photodiode array was originally useful for demonstrating channel electrophoretic analyses with dansylated amino acids.15,16,18 An alternative detection mode that uses cylindrical lenses and a highly sensitive CCD detector to preserve spatial resolution has been demonstrated for

(1) Kuhr, W. G. Anal. Chem. 1990, 62, 403R-414R. (2) Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 280R-314R. (3) St. Claire, R. Anal. Chem. 1996, 68, 569R-586R. (4) Gilman, S. D.; Ewing, A. G. J. Cap. Electrophor. 1995, 2, 1-13. (5) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1989, 246, 58-63. (6) Jankowski, J. A.; Tracht, S.; Sweedler, J. V. Trends. Anal. Chem. 1995, 14, 170-176. (7) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992, 64, 3045-3051. (8) Ewing, A. G. J. Neurosci. Methods 1993, 48, 215-224. (9) Swanek, F. D.; Sloss, S.; Ewing, A. G. Capillary electrophoresis for the analysis of single cells. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1997; Chapter 17. (10) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62, 1872-1876. (11) Bergquist, J.; Tarkowski, A.; Ekman, R.; Ewing A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12912-12916. (12) Swanek, F. D.; Chen, G.; Ewing, A. G. Anal. Chem. 1996, 68, 3912-3916. (13) Lillard, S. J.; Yeung, E. S.; McCloskey, M. A. Anal. Chem. 1996, 68, 28972904. (14) Lada, M. W.; Kennedy, R. T. Anal. Chem. 1996, 68, 2790-2797.

(15) Mesaros, J. M.; Luo, G.; Roeraade, J.; Ewing, A. G. Anal. Chem. 1993, 65, 3313-3319. (16) Mesaros, J. M.; Ewing, A. G. J. Microcolumn Sep. 1994, 6, 483-494. (17) Mesaros, J. M.; Ewing, A. G.; Gavin, P. F. Anal. Chem. 1994, 66, 527A537A. (18) Mesaros, J. M.; Gavin, P. F.; Ewing, A. G. Anal. Chem. 1996, 68, 34413449. (19) Gavin, P. F.; Ewing, A. G. J. Am. Chem. Soc. 1996, 118, 8932-8936. (20) Ewing, A. G.; Gavin, P. F.; Hietpas, P. B.; Bullard, K. M. Nature Med. 1997, 3, 97-99. (21) Liu, Y. M.; Sweedler, J. V. J. Am. Chem. Soc. 1995, 117, 8871-8872. (22) Liu, Y. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 2471-2476. (23) Liu, Y. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 3928-3933.

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kinetic analyses,21,22 parallel DNA separations,20 and a novel form of two-dimensional separations.23 Recently, we have expanded the types of analytes that can be studied by channel electrophoresis with the introduction of an electrochemical array detection scheme. Continuous channel electrophoretic separations of dopamine and catechol have been demonstrated in 21-µm-internal height channel structures using amperometric detection at a 100electrode, individually addressed array detector.19 The development of the electrochemical detector represents an important step toward our overall goal of monitoring dynamic neurochemical environments, since many neurotransmitters are easily oxidized in their native form, eliminating the need for either precapillary or on-line derivatization. Electrochemical detection in normal CE offers sensitivity comparable to that of fluorescence techniques, with detection limits approaching 10-19 mol.11 However, the detection schemes developed to date typically employ single, micrometer-size electrodes and are not directly applicable for detection in a 1-2-cm-wide rectangular channel. In addition, since spatial resolution across the channel width is required, a single electrode is not an ideal method of detection for channel electrophoresis. Spatially resolved, amperometric responses have been achieved in the channel electrophoresis format.19 Critical to the future success of the channel electrophoresis-electrochemical detection scheme is the ability to monitor concentration changes and/or repetitive mass changes in an environment. In addition, uniform responses from electrode to electrode are necessary for quantitative investigations, so real variations in analyte mass or concentration can be monitored as a function of time. The aim of the research described in this report is to characterize the detection scheme and demonstrate that changes in mass or concentration of easily oxidized species can be monitored. Electrode array detectors are evaluated in terms of the slope of calibration curves, linearity, and number of functional electrodes in order to evaluate present arrays and establish a baseline for future detector development. In addition, an approach is introduced that can be directly applied to circumvent nonuniform sensitivity within an electrode array. Finally, continuous separations in submicrometer internal height rectangular channel structures are detailed. EXPERIMENTAL SECTION Chemicals. Dopamine (3-hydroxytyramine) hydrochloride, 3,4-dihydroxybenzylamine hydrobromide, and MES (2-[N-morpholino])ethanesulfonic acid) were purchased from Sigma (St. Louis, MO). Stock solutions of analyte were made in 0.1 M perchloric acid and diluted in buffer solution to the appropriate concentrations. Buffer solutions were made in doubly distilled water and adjusted to the appropriate pH with solid sodium hydroxide. Buffers were filtered through 0.2-µm nylon filters (Supelco, Bellefonte, PA) prior to use. Sample Injection. Fused silica capillaries, 143-150-µm o.d., 10-15-µm i.d., and 70-76-cm length (Polymicro Technologies, Phoenix, AZ), were used for continuous sample introduction. The methodology used to fill sample introduction capillaries and to perform continuous injections and the safety considerations were similar to those previously described.19 The amount of analyte injected was calculated on the basis of the methods outlined for electrokinetic injection.24 (24) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272.

Channel Structures. Channel structures were formed from optical grade, ground, and polished fused quartz plates (Quartz Scientific Inc., Fairport Harbor, OH). The top quartz plate (4.84.9 cm long, 2-2.5 cm wide, and 0.23 cm thick) was beveled on one end to provide an angular entrance for the sampling capillary and on the opposite end to simulate end-column detection.25 The bottom quartz plate (7.5 cm × 7.5 cm × 0.23 cm) contained the electrode array, connection pathways, and contact pads directly microfabricated onto it. A small portion of the bottom plate was also beveled to accommodate the sampling capillary. Uniform glass microspheres (Duke Scientific, Palo Alto, CA) or liquid chromatographic particles mixed in UV-cure adhesive (Crosslink Technologies, Fort Wayne, IN) were applied only along the outer channel lengths and served to define the channel internal height as previously described.15,19 The channel structure with the microfabricated array detector (Figure 1) was placed across a quartz buffer reservoir (14 cm long, 4.5 cm wide, and 3 cm deep). Dow Corning high-vacuum grease was applied to prevent fluid leaks. A quartz wall was used to separate the individual reservoirs, each with a capacity in excess of 50 mL. The detection array was seated in the grounded portion of the buffer reservoir. The potential across the channel reservoirs was supplied by a Bertan high-voltage power supply (Hicksville, NY). Detection: Fabrication of an Electrode Array. The electrode array was fabricated in collaboration with the National Nanofabrication Users Facility (NNF) at Cornell University. The fabrication of an electrode array, which entails quartz plate preparation, metalization, photolithography, and etching of the metal layers, was performed at the NNF with standard photolithographic techniques. The electrode array pattern, designed in Symbad software (Cadence Design Systems, Inc.), consisted of 100 electrodes (95 µm wide × 1.2-2 mm long × 0.12 µm high) spaced across a 1-cm portion of the quartz plate, with individual connection pathways spanning the width of the plate and terminating at individual bond pads laid out for a dual-row 50-pin surface mount connector (P/N5653-050, InterCon Systems, Inc., Middletown, PA). The electrodes were made of evaporated platinum (100 nm) deposited on top of titanium (∼20 nm), which served as an adhesion layer. A 1.0-µm-thick layer of SiO2 was deposited across the connection pathways as insulation by plasma-enhanced chemical vapor deposition (PECVD). A “macromask” consisting of microscope slides was used to prevent deposition of SiO2 over the bond pads and electroactive portions of the electrodes during the deposition. Electrode Array Detection: Current Amplification System. Connection to each electrode in the array was achieved via a 2× 50-pin horizontal surface mount header (P/N5653-050, InterCon Systems, Inc.), physically attached to the quartz plate via two 0.045in.-diameter thru-holes. Electrical connection to the contact pad of each electrode was made via standard surface mount soldering techniques. The pin connector, and hence the array electrodes, were attached to the detection electronics via a 100-pin ribbon cable (4-in. length) with the appropriate 2- × 50-pin headers (InterCon Systems, Inc.). Amperometric detection was performed in the three-electrode format, with 100 individually addressed working electrodes. The electrochemical cell potential was supplied via a BAS CV-1B (Bioanalytical Systems, West Lafayette, IN) potentiostat, with all (25) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581.

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Figure 1. Channel electrophoresis with electrochemical array detection. The buffer-filled channel structure, with the electrode array directly fabricated onto the bottom plate, is suspended across two buffer reservoirs. A fused silica capillary and high-voltage power supply (not shown) are used to electrokinetically transport sample to the entrance of the rectangular channel. A high voltage is applied across the channel length to effect a separation. Five electrodes are shown spanning the width of the channel exit to simulate the 100-microelectrode array, individual pathways, and connections. A Pt auxiliary electrode and a Ag/AgCl electrode (both not shown) are also added to the detection reservoir to complete the three-electrode detection format.

working electrodes at the same potential. The working electrode circuit of the BAS potentiostat was not used. A 1-cm2 platinum electrode (Advanced Biosensor Technology, Yardley, PA) and a Ag/AgCl pellet electrode (World Precision Instruments, Sarasota, FL) were used as the counter and reference electrodes, respectively. The oxidation current at each of the 100 electrodes was monitored with electronics that were designed, developed, and built in-house. The voltage output from each of the 100 amplifiers was interfaced to a multifunction analog, digital, and timing input/ output board (AT-MIO-16, National Instruments, Austin, TX) via a system of seven, 16-channel multiplexers. The AT-MIO-16 was interfaced to a Micron Millennia P133 computer. Data Acquisition and Manipulation. Software programs to collect and manipulate data from each electrode in the array were developed and written in-house using LabView for Windows 3.1 (National Instruments). The individual electrodes in the array were hardware addressed, and hence data acquisition at each electrode was controlled digitally through software via the multiplexer addresses. The data acquisition rate was 2 Hz/100electrode scan. Fortner Transform 3.3 (Fortner Inc., Sterling, VA) was used for generation of two- and three-dimensional representations of the data. The data in Figure 7 have been 5-point smoothed with Transform. Microsoft Excel was used for some of the data manipulation and for generation of the plots shown in Figure 4. Calibration Curves. Experiments to determine the sensitivity of the electrode arrays were performed on the very first day of use. Briefly, the array detector was situated in the channel buffer reservoir, along with an Ag/AgCl reference and platinum counter electrodes. A 50-mL volume of analyte solution was added to the reservoir, and the oxidation current was monitored at +0.6 V vs Ag/AgCl. On average, at least 20 data points (10 s) were taken at each electrode for each concentration, and the average value over this period was used as the observed current for that 3840

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concentration. Four concentrations (6.11 µM, 0.0611 mM, 0.305 mM, and 0.611 mM) were used in the evaluation of electrode array 4. For electrode arrays denoted as 5 and 6, three concentrations were used: 0.607, 1.21, and 3.64 mM. The slope, intercept, and correlation coefficient of the calibration curves were obtained at each electrode, for each of the three arrays, with the average values for all functioning electrodes reported. Scanning Electron Microscopy (SEM). Images of channel structures and spacers were obtained with a JEOL JSM 5400 scanning electron microscope. The channel structures used in actual electrophoresis experiments were physically too large (3 in. × 3 in.) to fit in the instrument. Representative channels were made with similar quartz plates (without the electrode arrays) or standard microscope slides. The fabrication technique was the same for either electrophoresis experiments or SEM work. The liquid chromatographic stationary supports used in the work are commercially available from Supelco, Alltech (Adsorbosphere 100), and Micra Scientific. The size of the particles was either provided by the supplier or determined by SEM. The diameters of individual particles were measured, with 15-20 measurements per particle type. Construction and SEM evaluation of both the 3- and 0.6-µm channel structures have been performed at least twice. For quantitative measurements, errors are reported as standard deviations. RESULTS AND DISCUSSION Microfabricated Electrophoresis-Electrochemical Array Detection. Channel electrophoresis is a microseparation technique that can provide qualitative, quantitative, and time-based information for the investigation of dynamic microenvironments. Essential to the technique are a continuous sample introduction scheme, a wide, rectangular channel in which to perform electrophoretic separations, and a site-specific detector. The focus

Figure 2. Different analyte concentrations monitored with the microfabricated electrophoresis-electrochemical array detection scheme. (A, left) Plugs of dopamine (0.665 or 3.46 mM; 45 s duration) were injected from a sample vial into a fused silica capillary (10-µm i.d., 70-cm length) that was coupled to a buffer-filled rectangular channel (8-µm i.h., 4.8-cm length). The capillary and channel were filled with MES buffer (25 mM, pH 5.9) prior to the start of the experiment. The capillary and channel voltages used were 28 kV and 1500 V, respectively. The capillary was moved at a rate of 0.2 s/step. The separation current was 0.19 mA. Detection was at +0.8 V vs Ag/AgCl. (B, right) Three-dimensional plot of the electropherogram shown in (A).

of the work described here is on the array of 100 individually addressed electrodes that are used as an amperometric detection scheme. Examining Concentration Changes with Electrochemical Array Detection. The technical merits of channel electrophoresis for the investigation of concentration changes in a dynamic flow injection analysis (FIA) system18 and monitoring reaction kinetics of multiple components on line21,22 have previously been demonstrated. As we expect to achieve similar temporal characteristics, the ability to differentiate concentration changes of analytes with electrochemical detection is the key facet that must be demonstrated. To simulate concentration changes in a simple manner, four plugs of dopamine (45-s duration) have been injected into a buffer-filled capillary and allowed to migrate into the channel structure as the capillary is moved back and forth across the channel entrance. Figure 2A shows the results of an experiment where two different concentrations of dopamine (3.46 and 0.665 mM) were injected. In the experiment, the higher concentration, of dopamine is injected, followed by the lower concentration, and the pattern of injection is repeated. Similar results have been obtained with the reverse order of plug injection and 3,4dihydroxybenzylamine (DHBA; 0.77 and 3.08 mM, data not shown). Two different concentration regions are observed, regardless of the analyte or order of injection. In addition, the three-dimensional plot of this electropherogram, shown in Figure 2B, more clearly demonstrates the differences in sample concentration that have been detected with the array of electrodes. Although they are not the focus of the present investigation, the temporal characteristics of the plugs (i.e., duration) appear to be retained. Monitoring Mass Changes with Electrode Array Detection. Our first successful efforts with microfabricated electrophoresiselectrochemical detection have demonstrated the ability to achieve both continuous separations and long-term monitoring of analytes.19 Figure 3A shows the ability to detect repeated injections of dopamine at different levels with the electrophoresiselectrochemical array detection scheme. The mass injected with

electrokinetic methods is directly related to the time of injection.24 Thus, for a constant concentration of analyte, the amount delivered to a location along the channel entrance can be changed easily by the rate of sampling capillary movement. Hence, for the data in Figure 3, twice as much dopamine is delivered on outward steps (0.4 s/step, ∼600 fmol/electrode, as compared to the return step, 0.2 s/step, ∼300 fmol/electrode). From the three-dimensional plot of this electropherogram shown in Figure 3B, it is clear that, although two different masses have been detected, some variation in the response exists for the same amount of material injected across the different electrodes in the array. Thus, we have studied methods to calibrate and normalize electrode response. Linearity Studies with Electrode Arrays. The data in Figures 2 and 3 reveal that for either the same mass or concentration injected, the response of one electrode compared to that of its neighbor may be dramatically different. The variations in electrode response appear to be related to differences in the sensitivity of the different electrodes in the array. Calibration curves provide a means of evaluating the sensitivity of the individual electrodes in an array and might also reveal variations in detector fabrication. Figure 4 shows the slopes of individual electrodes obtained from linearity studies for three different electrode arrays. Three key features are apparent that might help explain the nonuniformity of response observed in channel electrophoresis experiments. First, not all 100 electrodes respond to changes in concentration, as indicated by a slope of zero. For the three arrays shown in Figure 4, the number of electrodes that respond to changes in dopamine concentration is between 79 and 88. Thus, there is a 12-21% failure rate in the fabrication procedure. As long as several electrodes adjacent to one another do not fail, this does not significantly affect the spatial resolution of detection. The average correlation coefficient for the electrodes in the three arrays is between 0.96 and 0.99. For the three electrode arrays, the average slope of the calibration curves varies over the range from ∼40 to 110 nA/mM dopamine. The second feature evident from Figure 4 is the large sensitivity differences among electrodes within an array. These Analytical Chemistry, Vol. 69, No. 18, September 15, 1997

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Figure 3. Mass changes monitored by variation of the capillary movement rate. (A, left) Two-dimensional representation of the data, showing the pattern of capillary movement and the differences in the detection currents. The sampling capillary (15-µm i.d., 75.5-cm length) with 7 kV applied across its length was moved at a rate of 0.4 s per 20.8-µm step on the forward pass and 0.2 s/step on the reverse. The channel was 4.85 cm long, with an 8-µm internal height, and a voltage of 1400 V was used. The separation buffer was MES (24.89 mM, pH 5.7). The capillary was electrokinetically filled with dopamine (3.85 mM). The separation current was 0.124 mA. Detection was at +0.8 V vs Ag/AgCl. The usable width of the channel was 1.1 cm. (B, right) Three-dimensional plot of the electropherogram in (A).

Figure 4. Plot of the slope obtained from calibration curves vs the 100 individual electrodes for three different electrode arrays. The slopes are normalized to the same electrode length.

differences appear to contribute to the cause of the nonuniform responses we have observed. Third, fabrication differences exist both within a batch of electrode arrays and from batch to batch. Figure 4 contains data from two electrode arrays that were made in the same batch and a third electrode array from a different batch. Since the data reported here were obtained on the first day of use of each of the arrays, some of the sensitivity differences might be inherent to the fabrication process. It is important to note that methods to either clean or pretreat electrodes made in a similar fashion have been developed.26 In one case, application of this method to an electrode array that had been used in 3842

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electrophoresis experiments over a 45-day period restored, on average, 76% of the initial response at each electrode. These preliminary results are promising and suggest that the pretreatment method should be used on a regular basis. Although these calibration studies do reveal some highly useful information and set a baseline for further improvement of both fabrication and experimental protocols, it should be noted that all the calibration curves have been generated under stagnant conditions and, therefore, do not directly mimic the flow environment in which the detector is used. Hence, the calibration data shown here must be used with the assumption that relative electrode responses are similar in stagnant and flowing conditions. Normalization of Array Response. To address the issue in a manner that is useful for both the present electrode arrays and future versions of the detection scheme, a normalization approach has been developed as a means to calibrate electrodes in the flowing electrophoresis environment. It accounts for differences in electrode response and provides a method to calibrate electrodes in subsequent experiments on a specific day. For example, the three-dimensional plot of an electropherogram shown in Figure 5A for a 31-electrode portion of the data in Figure 3 demonstrates that normalization can provide a nearly uniform response. The maximum responses on both the forward (from electrode 27 toward 61) and reverse (from electrode 61 toward 27) passes have been used to generate a normalization factor for each electrode in the array for the particular amount injected. The results of the process can be observed more clearly in Figure 5B, where a comparison of the 31 electrodes before and after correction is shown. The two different magnitudes represent the responses of 600 and 300 fmol/electrode, respectively, with the dashed and solid lines representing raw and normalized data for each mass. The normalization approach provides more uniform (26) Josowicz, M.; Janata, J.; Levy, M. J. Electrochem. Soc. 1988, 135, 112-115.

Figure 5. (A, left) Three-dimensional plot of the electropherogram for a 31-electrode portion of the data shown in Figure 3B, normalized to the maximum response. (B, right top) Comparison of the raw and normalized data at the 31 electrodes for the two different masses injected. (C, right bottom) Extract of a linear electropherogram from electrode 42 from the data in (A).

responses from electrode to electrode, mimics experimental conditions, and does not require any off-line calibration experiments. This allows the method to be used to monitor relative concentration dynamics. Importantly, subsequent data series on a particular day of experimentation can be normalized with the factors generated on the first pass, as has been done with the second forward and reverse passes of the data from Figure 3 and shown in Figure 5A. Finally, the linear electropherogram extracted from electrode 42 (Figure 5C) demonstrates that repeated sampling of different amounts of material for a given concentration can be determined. Integration of the area under the peaks at each of the 31 electrodes in Figure 5A reveals an average ratio of 2:1, as expected for the two different mass quantities. Additionally, the peak height ratio for the electrodes for the two different masses injected is 2. Relative quantitation can be accomplished on the basis of either peak area or peak height by this method. Submicrometer Internal Height Channels. Miniaturization of the internal height (i.h.) of the channel has been critical to our success in the development of the electrochemical detection scheme. Here, using the glass microsphere adhesive technique, channel structures with 8-µm i.h. have been routinely used in electrophoresis experiments. The smaller channel internal height has allowed us to perform continuous separations with ∼300V/ cm applied across the channel length20 and lower our detection limits by a factor of 2 vs the 21-µm-i.h. channels.19 In addition, the coulometric efficiency (mass detected/mass injected) of the detection scheme increases by ∼20% with the use of smaller

internal height channels. Taken together with the methods to improve sensitivity via electrochemical pretreatment, further miniaturization of the internal height should result in additional improvements in our detection scheme. Uniform glass microspheres have routinely been used to define the separation space for channel structures with internal heights from 8 to 109 µm.15-23 However, they have not been useful for defining channels with internal heights of