Microfabrication of a Planar Absorbance and Fluorescence Cell for

Chem.19966861040-1046 ... excitation with a 20-fold improvement in signal-to-background levels due to reduced scattering, utilizing a form of sheath f...
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Anal. Chem. 1996, 68, 1040-1046

Microfabrication of a Planar Absorbance and Fluorescence Cell for Integrated Capillary Electrophoresis Devices Zhenhua Liang, Nghia Chiem, Gregor Ocvirk, Thompson Tang, Karl Fluri, and D. Jed Harrison*

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

A micromachined absorbance and fluorescence detection cell for application to capillary electrophoresis within planar glass substrates (chips) is described. A microfabricated U-cell for absorbance provides a longitudinal path 120-140 µm long parallel to the flow direction and gives at least a 10-fold increase in absorbance compared to an absorbance path transverse to the flow direction. Absorbance detection limits of 0.003 AU gave ∼6 µM detection limits for hydrolyzed fluorescein isothiocyanate dye. The same device can be used for longitudinal fluorescence excitation with a 20-fold improvement in signal-tobackground levels due to reduced scattering, utilizing a form of sheath flow. Fluorescence detection limits of ∼20 000 molecules and 3 nM were obtained for fluorescein. Obtaining good limits of detection when using optical detection has been a challenge in capillary electrophoresis (CE), due to the short path lengths engendered by the small capillary diameter.1-3 Fluorescence detection has proven to be very effective.4-6 Nevertheless, absorbance detection remains more generally accepted due to its wider applicability. Several methods, including the use of Z-cells,3,7,8 as in liquid chromatography detection,9 or widening of the capillary at the detection point, can improve detection limits by increasing the optical path length. With micromachined CE (µ-CE) devices etched on planar glass plates, the path length problem is equally severe, so to date, fluorescence detection has been the principal optical detection method employed.10-14 (1) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135-143. (2) Bruno, A. E.; Gassmann, E.; Pericles, N.; Anton, K. Anal. Chem. 1989, 61, 876-883. (3) Bruin, G. J. M.; Stegeman, G.; Van Austen, A. C.; Xu, X.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1991, 559, 163-181. (4) Green, J. S.; Jorgenson, J. W. J. Chromatogr. 1986, 352, 337-343. (5) Zare, R. N.; Gassmann, E. Eur. Pat. Appl. EP 21660, 1987. (6) Cheng, Y.-F.; Dovichi, N. J. Science 1988, 242, 562-564. (7) Chervet, J. P.; Van Soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-449. (8) Moring, S. E.; Reel, R. T.; Van Soest, R. E. J. Anal. Chem. 1993, 65, 34543459. (9) Stevenson, R. L. In Liquid Chromatography Detectors; Vickrey, T. M., Ed.; Chromatographic Science Series 23; Dekker: New York, 1983; Chapter 2, pp 23-86. (10) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (11) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (12) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (13) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 28582865.

1040 Analytical Chemistry, Vol. 68, No. 6, March 15, 1996

Micromachining allows complex capillary geometries to be formed so that optical components may be integrated into a planar device.10 In this way, micromachined Z-cells can be fabricated, as was demonstrated by Verpoorte et al.15 in the realization of an absorbance Z-cell for liquid chromatography detection. That device used the crystal planes of silicon(111) to form mirror planes to reflect a beam from above the planar device into the capillary. CE devices are not readily fabricated in silicon due to the high voltages usually employed and the conductivity of Si.16 Unfortunately, mirror planes are not easily formed in amorphous glass since it etches isotropically,17 so an alternate strategy to launch and collect light is required. In this report, we describe the fabrication of a planar optical U-type cell in glass and its application for both fluorescence and absorbance detection. The cell provides a 10-fold improvement in absorbance detection limits by probing the capillary along the longitudinal rather than the transverse direction. It also enhances fluorescence detection limits, since the fluorescent signal may be detected well away from the point at which the light enters the capillary. This dramatically reduces the amount of scattered light collected, resulting in an improved signal-to-background ratio. CELL DESIGN Figure 1 shows the layout and dimensions of a µ-CE device with integrated optical components. Reservoirs A-C connect to the electrophoresis injector and separation capillaries, which form a U-shaped cell with a 100-140 µm longitudinal path length. Reservoirs D and E connect to channels into which optical fibers were inserted to launch and collect light. These two reservoirs were designed for introducing index matching fluid to reduce scattering and reflection losses. The configuration used does not readily allow for lenses at the launch and collection points, although some shaping of the fiber tips to form lenses can be achieved by etching the optical fibers. To ensure that the light launched stays within the separation channel and is all collected, a launch fiber with small numerical aperture (NA) and small core diameter should be used. For a single-mode fiber, the beam waist, w, at the exit point is (14) Jacobsen, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (15) Verpoorte, E.; Manz, A.; Lu ¨ di, H.; Bruno, A. E.; Maystre, F.; Krattiger, B.; Widmer, H. M.; van der Schoot, B. H.; de Rooij, N. F. Sens. Actuators 1992, B6, 66-70. (16) Harrison, D. J.; Glavina, P. G.; Manz, A. Sens. Actuators 1993, B10, 107116. (17) Fan, Z.; Harrison, D. J. Anal. Chem. 1994, 66, 177-184. 0003-2700/96/0368-1040$12.00/0

© 1996 American Chemical Society

Figure 2. Geometry of channels and collection fibers around the U-cell of the flow channels. Flow channels are etched into the bottom plate only, and a top plate is thermally bonded.

Figure 1. (a) Geometric layout of channels etched with glass plate. Letters mark reservoir access points. (b) Blowup of U-cell used for detection. Dimensions in millimeters.

given by18,19

(

w ) d 0.65 + V)

)

1.619 2.879 + V1.5 V6

(1)

dπ NA λ

(2)

where d is the core diameter and λ is the wavelength. For a 3.1 µm core, single-mode fiber with an NA of 0.1 and a wavelength of 488 nm, the waist is 3.93 µm at the launch point. The angle of dispersion is given by

θ ) arcsin(NA/n)

(3)

where n is the refractive index of the transmitting medium. The beam diameter, w′, some distance x from the launch point, can be estimated as

w′ ) w + 2x tan θ

(4)

if traveling in a single medium. For a beam launched into water from a 3.1 µm fiber with an NA of 0.1, the beam diameter will be ∼22 µm after passing through a 120 µm long cell. Such a beam would be essentially contained within a 20 µm deep channel, which would be a trapezoid 50 µm wide at the top and 20 µm wide at the bottom.

Since the launch fiber cannot be exactly at the detection cell edge due to fabrication constraints, there will be additional beam expansion. Assuming a 50 µm separation between the aqueous sample channel and the fiber tip, and assuming that the value of n for this medium matches that of the glass chip, n ) 1.519, gives a beam width of 10.5 µm at the cell-glass interface. Assuming that Snell’s law governs the refraction angle at the glass-water interface, and ignoring any lensing effect of the concave lens formed by the curved glass walls, the beam will expand to 28 µm after traversing the 120 µm cell and will be ∼32 µm in diameter at the collection fiber 50 µm from the cell wall. Larger, multimode launch fibers could be used to increase light input. Assuming the same conditions as above, a beam launched from a 10 µm fiber expands to 35 µm across the cell and to 41 µm at the collection fiber. A 20 µm launch fiber gives a beam 45 and 51 µm in diameter at the same two points. Consequently, a 50 µm collection fiber is adequate, but larger cross-section flow channels are needed for larger launch fibers if a 120 µm path length is used. (We note that the angle of acceptance by the collection fiber is also given by eq 3, so with the same numerical aperture, a 50 µm fiber will efficiently collect the light.) Alignment of the optical fiber core with the separation capillary can be achieved in several ways. Figure 2 illustrates the required geometry, in which the center of the channel for the optical fiber is offset from the plane of the glass-glass bond, so that the light path is aligned with the center of the separation channel. This was accomplished by etching the fiber channel after bonding the cover plate to the etched plate, as described later. The cell volume makes a contribution to band broadening. Using the standard formula,20 assuming a rectangular cell volume gives the height equivalent to a theoretical plate contributed by the detector cell, Hdet, as

Hdet ) (18) (a) Barnoski, M. Fundamentals of Optical Fiber Communications, 2nd ed.; Academic Press: New York, 1991. (b) Adams, M. J. An Introduction to Optical Waveguides; J. Wiley and Sons: Chichester, 1981; p 276. (c) Hunsperger, R. G. Integrated Optics: Theory and Technology, 2nd ed.; Springer-Verlag: Berlin, 1991; Chapter 6. (d) Sharma, A. B.; Halme, S. J.; Butusov, M. M. Optical Fiber Systems and Their Components; SpringerVerlag: Berlin, 1981; Chapters 1, 2. (19) Adams, M. J. An Introduction to Optical Waveguides; J. Wiley & Sons: New York, 1981; p 276.

l2 12did

(5)

where l is the cell path length and did is the injector-to-detector distance. A path length of 120 µm will contribute 0.03 µm of plate height for the device studied here and as little as 0.02 µm for (20) Sternberg, J. C. Adv. Chromatogr. 1966, 2, 206-270.

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typical devices reported recently,12 for which did ≈ 5 cm. Given the plate height range of 0.2-2 µm that has been reported, a detector length of up to 250 µm would introduce additional band broadening of 50-5%, respectively. EXPERIMENTAL SECTION Device Fabrication. A photomask glass used for photomasks was patterned photolithographically and etched with an HF/HNO3 mixture, as described previously.17 Holes (1.5 mm diameter) were drilled in a cover plate with a diamond drill bit, and the two plates were then cleaned using a Model 2066 high-pressure cleaning station (MicroAutomation) under a class 100 clean hood. The plates were bonded by heating at 595 °C for 6 h. Before bonding, the channels were etched either 20 µm deep and 50 µm wide with a transverse cell path length of 140 µm, or 10 µm deep and 30 µm wide with a cell path length of 120 µm. Near reservoir C, the channel was expanded to 200 µm wide. The optical fiber channels were enlarged after bonding the top plate by pumping 2-5% HF solution under 1.5 psi pressure for about 5-6 h. (Extreme care must be taken to avoid exposure to the HF solution.) This expanded the original 50 µm × 20 µm trapezoidal channel to about 230 µm wide by 150 µm deep, giving an oval shape. The walls between the fiber and separation channels were reduced to ∼10 µm thickness. Optical fibers with nominally 125 µm diameter glass cladding, 250 µm diameter jacket, and a core of 3.1 (F-SA), 7.9 (F-SS), or 50 (F-MSD) µm diameter were from Newport. Fibers were cleaved before use with a Newport F-BK2 fiber cleaver. The optical fibers were stripped of their polyimide coating and then etched in an HF/HNO3 mixture (10:1, using 49% HF and 70% HNO3). This reduced the outer cladding diameter slightly, to allow insertion into the fiber channel. Because the silica cladding etches faster than the core, the 3.5-4 min etch time created a rounded protuberance of core silica, giving a slightly convex lens at the fiber tip.21 The fibers were inserted into the fiber channels in a class 100 clean hood, with the assistance of a microscope and a fiber positioner (Newport FP-1 positioner mounted on Newport 423 translation stages). Caution is required to avoid damaging the fiber during insertion. After insertion, index matching fluid, 1,4dibromobutane (Aldrich, n ) 1.519), was introduced through reservoirs D and E. The entry points for the fiber were then sealed with epoxy resin (Araldite 5 min epoxy), and the reservoirs were sealed with parafilm. Reagents. The Na+ salt of fluorescein (Molecular Probes, Eugene, OR), fluorescein-5-isothiocyanate (FITC; Sigma, St. Louis, MO), and concentrated HF (Fisher, reagent grade) were used as received. The running buffer for all electrophoresis was 20 mM boric acid/100 mM tris(hydroxymethyl)aminomethane adjusted to pH 9.0. All fluorescein and FITC solutions were prepared in this same buffer to ensure identical ionic strengths and to avoid sample stacking effects. FITC solutions were allowed to hydrolyze overnight before use. Polymer waveguides were formed using Norland Adhesive, UV-curable types 71 and 88, or Epo-Tek 2 part optical adhesive (Epoxy Tech., Billerica, MA). Glass used for photomasks was obtained from AGFA Gevaert (Belgium) (73% SiO2, 13.5% Na2O, 1.8% Al2O3, 8.9% CaO, and 2.7% MgO by X-ray fluorescence). (21) Kayoun, P.; Puech, C.; Papuchon, M.; Arditty, H. J. Electron. Lett. 1981, 17, 400-401.

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Figure 3. Schematic of various detection geometries around the microstructure (µ-TAS). 1, Launch fiber; 2, fluorescence excitation lens; 3-5, fluorescence or transverse absorbance detection with lens, pinhole, and filter, respectively; 6 and 7, longitudinal absorbance collection fiber and filter, respectively; 8, transverse absorbance source using lens.

Instrumentation. The computer-controlled power supply and relay arrangement has been described previously.17,22 Hamamatsu photomultiplier tubes (PMTs) were used to measure the fluorescence and absorbance signals. Collection optics used were 25× Leitz Fluotar (0.35 NA) or 7× Rolyn (0.2 NA) objectives. The optical arrangements for detection are illustrated in Figure 3. The Uniphase Cyonics 488 nm laser was operated at 5.86 mW output power, measured with a Newport Model 835 power meter. The laser power was coupled into a 3.1 µm fiber using a single-mode fiber coupler. The output power of the fiber was 0.143 µW. A Gaertner L125B ellipsometer was used to determine the refractive index of the crown glass at 633 nm. Procedures. Devices were first filled with buffer, and then a sample dye was introduced at reservoir B. Sample was injected with a potential applied between reservoirs B and C (effective length, 6.5 cm). Separation was performed with a potential applied between reservoirs A and C (effective length, 5.8 cm). Here, the effective length of the 200 µm wide segment near reservoir C is expressed as its equivalent 0.15 cm length of 30 µm wide channel.23 The distance from injection to detection point, did, was 3.7 cm. Several optical detection geometries were employed. Transverse absorbance involved illumination from below the chip (element 8 in Figure 3) with laser light delivered by mirrors and collection above the chip with a 25× objective (5, Figure 3) and a 200 µm pinhole at the image plane. A 488 nm notch filter was used for collection, and a 1% neutral density filter was used to (22) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 2637-2642. (23) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 34853491.

Figure 4. Scanning electron mirograph (SEM) of device cut and polished to display the launch and collection fiber channels and the U-cell optical path. Some SEM sample preparation damage is visible below the flow channel. Flow channel was 20 µm deep.

avoid photobleaching. Transverse fluorescence, our conventional detection method, was performed with excitation by laser light delivered by mirror and a lens at 45° to the upper surface normal (2, Figure 3). Emitted light was collected with a 7× objective, a 200 µm pinhole, a 508-533 nm bandpass filter (5, Figure 3), and a PMT. Longitudinal absorbance was performed by launching light into a 3.1 µm single mode fiber (1, Figure 3) and collecting it with a 7.9 µm optical fiber, unless otherwise indicated (6, Figure 3). A 488 nm notch filter was also used. Longitudinal fluorescence was performed using the 3.1 µm launch fiber for excitation and a 7× objective, a 200 µm pinhole, and a 508-533 nm bandpass filter (5, Figure 3) for collection. The detector was positioned about midway along the 120 or 140 µm optical cell path. RESULTS AND DISCUSSION Fabrication. The micromachined optical cell was fabricated in crown glass using conventional photolithography and chemical etching. The procedure described above, etching the fiber channel after bonding the cover plate, results in a fiber channel with a center that is offset from the bonding plane of the two plates. This aligns the fiber core with the center of the separation channel, as sketched in Figure 2. Figure 4 shows a side view of the assembled structure, in which the curvature of the walls is apparent. This curvature limits the approach of the fibers to the wall separating the fluid and fiber channels, as seen in the top view in Figure 5. The alignment of the fiber cores with each other is quite satisfactory, as seen in Figure 5. However, the fiber’s positioning relative to the fluid channel is slightly shifted. This problem could be resolved by redesigning the etch mask to shift the channel positions slightly. Alternatives to the fabrication procedures used here are possible. For example, the channels could be etched in top and bottom plates, although this requires careful alignment to ∼1 µm during bonding and the use of a two-mask process to define channels with two different depths. Another possibility is to form the optical waveguides in the glass by ion exchange doping rather than using conventional optical fibers, but this requires a waveguide that could survive the subsequent top-to-bottom plate bonding, which is usually done at elevated temperature. We did attempt

Figure 5. Optical micrograph showing launch of 488 nm beam from lower fiber. (A) Beam path illuminated by fluorescein at 520 nm (503533 filter) and (B) scattering at center wall and exit points seen with a 488 nm filter. Flow channel was 20 µm deep, with a 140 µm longitudinal optical path length. Table 1 absorbance path (length, µm) longitudinal (140) fiber combination (launch/collection)c 3.1/50 3.1/7.9 3.1/3.1 transverse (20)

fluorescein concn (µM)

62.5 62.5 62.5 12.5 62.5 625

absorbance (AU) theora measd

0.056 0.056 0.056 0.0112 0.008 0.080

efficiencyb (%)

0.0387 0.0548 0.0565 0.0112 noised 0.012

69 98 101 100 64

a Calculated from A ) bC, where b is path length and  ) 6.4 × 104 M-1 cm-1. b Ratio of measured to theoretical absorbance. c Diameters of the launch and collection waveguides (µm). d Too little signal to be distinguished from noise.

to fill the channels with polymer-based waveguides, but the volume change upon curing resulted in numerous scattering defects in the waveguide. Optical Cell Performance. The efficiency of the optical absorbance cell was tested using several combinations of launch and collection fibers. The entire flow path was filled with a steady stream of fluorescein solution, and the absorbance was measured at 488 nm in a cell with a 140 µm longitudinal path length. The data are presented in Table 1. Combining a 3.1 µm launch fiber with either a 3.1 or 7.9 µm collection fiber gave the theoretically predicted absorbances, within (2%. Larger collection fibers resulted in larger PMT signals due to increased light collection, Analytical Chemistry, Vol. 68, No. 6, March 15, 1996

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Figure 6. Simultaneous longitudinal absorbance and fluorescence detection of 50 µM FITC, pH 9.0 at 2 kV applied (Vid ) 1.28 kV).

but the cell’s absorbance efficiency, defined here as the ratio of observed to theoretical absorbance, was decreased. This is apparently due to stray light effects resulting from scattered 488 nm light that reached the collection fiber without traveling entirely through the sample solution. In this sense, the theoretical calculation of suitable collection fiber diameter proved inadequate. A collection fiber of 7.9 µm was used for the studies described below. A transverse absorbance path was also studied (elements 3, 4, 5, and 8 in Figure 3), using a cell etched 20 µm deep. The absorbance efficiency of this geometry could not be improved above 64%, indicating strong stray light effects. Consequently, the 140 µm long longitudinal path gave a net 11-fold increase in absorbance, even though only a 7-fold increase would be expected on the basis of the path length difference. The calculations presented above predict that the beam diameter for the 140 µm path in the cell of Figure 5 should be about 30 µm where it exits the U-cell. However, due to the position of the cell off to one side, the beam is truncated by the contacting wall and exits with about a 22 µm width. At the initial point of contact with the wall, the beam has traveled 50 µm in water, at which point eq 4 predicts an 18 µm diameter. Measurement of the beam width in Figure 5A at this point gives 18 ( 2 µm, in good agreement with theory. Figure 5A also illustrates that the beam is larger than the 7.9 µm collection fiber, leading to some losses. Further losses clearly occur from scattering at the flow channel-glass interface, as seen using a 488 nm bandpass filter, Figure 5B. However, the index matching fluid minimizes scattering at the other material interfaces. It should be noted that losses in transmittance, which are independent of sample concentration, are not a serious problem unless the incident intensity is very low, whereas stray light effects will drastically reduce the cell performance. Figure 6 shows simultaneous determination of hydrolyzed FITC by longitudinal absorbance detection and longitudinal fluorescence excitation in a 120 µm long cell. The separation efficiencies varied linearly with applied potential as expected (0.5-5 kV) but were the same for both longitudinal fluorescence and absorbance measurements at each potential (e.g., 19 000 plates at 5000 V applied, Vid ) 3190 V, data not shown). Separation 1044

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Figure 7. Plot of normalized absorbance or concentration versus the calculated length of the injected sample plug for longitudinal (b) absorbance and (+) fluorescence measurements, with 50 µM fluorescein, pH 9.0. Also plotted is the theoretical normalized concentration obtained by numerically solving eq 7. The solid curves are for clarity only; the dashed curve shows the expected normalized absorbance in the absence of any disperson effects. Electrophoresis performed with Vsep ) 3 kV, tm ) 65.6 s.

efficiency was also measured with the previous, transverse fluorescence detection configuration we employed, bringing the laser beam in from outside the chip at about 45° to the collection optics, and observing a 30 µm length of channel. This resulted in the same efficiency as obtained with longitudinal absorbance at a given potential, confirming that the larger cell length of the absorbance detector (120 vs 30 µm) did not increase band broadening. Dispersion Effects. Dispersion due to diffusion plays a significant role in determining concentration at the detection point. This issue was examined in detail for the longitudinal cell. For the low linear velocities involved (j1 mm/s), the dispersion of an extended sample plug can be described by an error function expression given by Crank:24

(

C(x) ) 1/2Co erf

h-x 2xDdid/u

+ erf

h+x 2xDdid/u

)

(6)

where Co is the original concentration, 2h is the initial length of the plug, D is the diffusion coefficient, did is the injector-to-detector distance, u is the linear velocity in the capillary, and x is the longitudinal distance from the center of the absorbance cell. If the sample plug length is short enough relative to the optical cell path length, dispersion will result in a nonuniform concentration gradient within the cell. In this case, the Lambert-Beer law must be expressed as

A)

∫

+a

C(x) dx

(7)

-a

where 2a is the width of the optical cell. Equations 6 and 7 are readily solved numerically using the Hi-Q software package (National Instruments, Austin, TX). The results of this dispersion (24) Crank, J. The Mathematics of Diffusion, 2nd ed.; Clarendon Press: Oxford, 1975; pp 14-16.

calculation are plotted as a function of plug length in Figure 7, for a detection cell length of 2a ) 120 µm, D ) 3.3 × 10-6 cm2/s (determined at pH 8.5 by polarography at 20 °C), did ) 3.7 cm, and a linear flow velocity of 0.056 cm/s. (Velocity was calculated from the applied separation field (3 kV across 5.8 cm) and the measured mobility of 1.09 × 10-4 cm2/V‚s.) Injected plug lengths were varied by controlling injection time and potential and were calculated from

2h )

tinjVinj µ 6.5 cm

(8)

where µ is the mobility, determined from migration times measured as a function of applied field. We have previously shown that diffusion effects provide the main source of band broadening in the microchips, once sample plug size and detection volume are accounted for.10,17,22,23 Those results confirm that diffusion occurs in the channels with coefficients similar to values measured by other means. In the present experiments, the running buffer and sample solutions were prepared from the same stock buffer, so that sample stacking phenomena should be insignificant. Under these conditions, the solution to eqs 6 and 7 should describe the dispersion in peak height seen with varying sample plug size. Figure 7 shows how the peak absorbance and fluorescence varied with plug length, compared to the calculated effect of dispersion. The data exhibit lower signal than that calculated around the knee of the curve, but the theoretical trend matches the observed trend. The discrepancy is likely due to some inaccuracy in the diffusion coefficient due to differences in temperature and solvent conditions between the electrophoretic and polarographic measurements. For short plug lengths, diffusion at the injector has been shown to increase the amount injected over the calculated lengths.17,22,23 Absorbance Detection Limits. The data in Figure 7 illustrate that varying the plug length allows control of the peak absorbance. We used this feature as a convenient means to vary absorbance of 50 µM plugs of fluorescein and thus to obtain the absorbance detection limit for a cell with a 120 µm path length. A plot of signal-to-noise (S/N) ratio versus absorbance, obtained by decreasing the injected plug lengths of fluorescein, was linear, with a slope of 990 and an intercept of -0.6 (R2 ) 0.988). The S/N ratio reached 3 at about 0.003 AU. The absorbance detection limit obtained is rather high. However, it was limited by the poor coupling into the optical fiber that we achieved (>10 000:1 loss), and by the use of an Ar+ ion laser source. Use of a stabilized source and better coupling would improve detection limits with the same cell design. Concentration detection limits were measured by varying the concentrations of FITC and fluorescein for a fixed injection plug length. The absorbance calibration curve for the earliest eluting peak of hydrolyzed FITC was linear between 20 and 100 µM formal concentration of FITC, measured in a 120 µm path length cell. The S/N ratio increased linearly with concentration of FITC for plug lengths of 800 µm, with S/N ) 9.1 ( 0.5 at 20 µM FITC and a slope of 0.18. The concentration detection limit, in terms of formal injected concentration of FITC, was estimated to be about 6 µM. Dispersion due to diffusion (a factor of ∼0.8, vide supra) as well as the distribution of FITC between several hydrolysis products means the true concentration detection limit was somewhat lower.

Figure 8. Fluorescence detection of 10 nM fluorescein, pH 9.0, with Vsep ) 3 kV and transverse fluorescence excitation using (a) 3.5 mm injection plug length (20 s at 1 kV), (b) 0.8 mm plug (5 s at 1 kV), and (c) 0.35 mm injection plug (1 s at 2 kV). This is contrasted with longitudinal fluorescence excitation using (d) 0.35 mm injection plug (1 s at 2 kV).

The absorbance calibration curve was also determined by varying fluorescein concentrations in a 140 µm longitudinal path length cell. Concentrations between 7 and 300 µm were studied, giving a calibration slope of 750 M-1 and an intercept of -3 × 10-3 AU for a sample plug length of 800 µm. The R2 factor was 0.998. A plot of S/N versus concentration was also linear. Extrapolation to S/N ) 3 gave a concentration detection limit of 5.6 µM for fluorescein. Fluorescence Detection. Scattering of the excitation light creates a background that can raise the detection limits in fluorescence detection.6 Within the planar devices we have used, there is a fair amount of scattering from the glass and from the curved walls of the channels. The fiber launched design we employed here for absorbance can also be used for fluorescence excitation. In this case, the beam is launched longitudinally along the channel, and collection optics can be located at 90° to the beam. Figure 5 shows that the light beam is smaller than the channel it is launched into, so the solution forms a sheath around the beam within the cell. This sheath geometry is similar to the surrounding water sheath in a sheath flow fluorescence cell, except the sample solution itself forms the “sheath”. The sheath flow cell provides good detection limits, since there is little light scattering from the solvent “cell walls”. For our cell design, the same sheath effect will occur so long as detection is performed away from the scattering at the entry and exit points of the cell. This can result in improved detection limits. Figure 8 shows the response to 10 nM fluorescein for transverse fluorescence excitation versus longitudinal excitation by the fiber. A 7× objective with a 200 µm pinhole was used for detection in both experiments, giving an observation zone about 30 µm long. For transverse excitation, an injection time of 20 s at 1 kV produced a measurable signal with S/N ) 7.2. An injection for 1 s at 2 kV or even 5 s at 1 kV produced no detectable signal for the transverse excitation. In contrast, a peak with S/N ) 5.0 was seen when a 1 s injection was used at 2 kV and longitudinal excitation. As discussed above, the longer injection plug reduces the effect of dispersion on concentration, so this result indicates an improved detection limit. This improvement is even more remarkable considering that the laser power was unchanged, but the coupling efficiency to the fiber reduces the Analytical Chemistry, Vol. 68, No. 6, March 15, 1996

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photon flux more than 10 000-fold for the longitudinal excitation. Better coupling efficiency is possible, especially if a 5 or 8 µm launch fiber is used. This is expected to result in further improvement in detection limit for longitudinal excitation. Use of a higher numeral aperture collection lens can also improve detection limits, though this will be true for both transverse and longitudinal excitation. Given the dispersion which occurs due to diffusion, the calculated curve in Figure 7 indicates that the maximum concentration of the peak in Figure 8d is about 5 nM, so the mass detected in Figure 8d corresponds to about 50 zmol (∼30 000 molecules). The S/N ratio was 5.0 in Figure 8d, so a linear extrapolation to 3 indicates concentration and mass detection limits of 3 nM and about 20 000 molecules, respectively.25 As may be seen in Figure 8, the S/N ratio is poorer for longitudinal excitation than for the transverse geometry, due to the lower excitation beam intensity. However, the ratio of sample-to-background signal for longitudinal excitation was about 20 times higher than that for transverse excitation. This difference confirms that the improved detection limit arises from better discrimination against scattered light. CONCLUSIONS This study shows that it is possible to microfabricate an absorbance cell in glass, without the use of integrated mirrors or collection optics, if the cell path length is relatively short. A considerable increase in path length compared to transverse passage of the micromachined flow cell by the probe beam is obviously possible. The tests were performed with a single-mode fiber and a laser source. However, calculations presented here clearly show that this approach can be adapted to multimode fibers, and there is, of course, no restriction that a laser be used as the source. Consequently, a generally applicable absorbance cell design has been demonstrated. While improvements in (25) The cylindrical, longitudinal detection volume was estimated using the 20 µm beam diameter shown in Figure 5 and the 30 µm spot size observed by the microscope.

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Analytical Chemistry, Vol. 68, No. 6, March 15, 1996

absorbance detection limits can still be made by using a stabilized light source and better electronic detection circuitry for the photomultiplier tube, the basic principles of the design have been established. The ability to use absorbance detection for on-chip electrophoresis will make the devices useful for a broader range of analytes. Fluorescence detection using the longitudinal excitation geometry offered by the present chip design offers lower background signal by reducing scattering effects. This gives better detection limits, which will be a key factor in applying on-chip electrophoresis to many biological assays in which concentrations are low. Further improvements in performance can be achieved with minimal effort by using more highly efficient collection lenses, larger diameter excitation fibers, and more efficient coupling of the source into the fiber. The present design realizes its advantage in a manner similar to that used with sheath flow detection; however, the surrounding sheath in the chip design is, in fact, the sample solution itself. This factor may reduce the complexity of detection cell design compared to sheath flow, since an additional pump and solvent stream are not needed. At present, the insertion of fibers into the chip provides a simple means of coupling the fibers to external sources and detectors. However, integrated waveguides may be fabricated on the chips, using methods compatible with the channel fabrication, and this will lead to devices that are even simpler to fabricate. ACKNOWLEDGMENT We thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for support of this work. Z.L. is grateful to NSERC for an International Fellowship. We thank the staff at the Alberta Microelectronic Centre for their assistance. Received for review August 1, 1995. Accepted December 19, 1995.X AC950768F X

Abstract published in Advance ACS Abstracts, February 1, 1996.