Anal. Chem. 1995, 67, 3367-3371
Spatial-Scanning Laser Fluorescence Capillary Electrophoresis
for
Stephen C. Beale* and Sara Jane Sudmeier Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294
A laser-induced fluorescence (LIF) detector using epiillumination and confocal optical detection geometry is described. The LIF detector is designed to scan the entire length ofthe separation cap-. The capiby is mounted on a precision translational stage which moves the entire capillary through the probe beam. The design of the laser scanner and the results from optimization experiments are presented. The LIF scanner can be used to monitor fluorescence from fluoresceinisothiocyanate-labeledproteins focused by capillary isoelectric focusing or to follow the time course of a separation. Dynamically changing the effective separation length is shown to offer a means to decrease analysis time. A method for directly measuring the diffusion coefficient is also presented. Laser-induced fluorescence (LIF) detection offers a highly sensitive means for detecting migrating zones in capillary electrophoresis (CE). Laser fluorescence systems have provided mass detection limits at subattomole levels' and concentration detection limits substantially lower than those obtained by W-vis detection. The capability to monitor the progress of the separation process or to dynamically alter the length of the separation bed by scanning the entire capillary offers several advantages over conventional instrumentation. Separation time can be optimized since the duration of the run need only be long enough to resolve the components of interest. Thus, sample throughput will be increased as the solutes do not need to migrate through the entire length of the separation bed. The ability to scan the capillary will facilitate the measurement of the solute d ~ s i o ncoefficient, from which hydrodynamic radius, solvent viscosity, and expected peak variance due to diffusional band-broadening can be calculated independent of electrophoretic contributions to peak width. Epi-illumination confocal laser fluorescence detection has recently become a popular means to achieve sensitive LIF dete~tion.2-~Laser confocal fluorescence systems have been successfully used for the detection of DNA fragments separated in a capillary array5,6s8or on microfabricated chipsg and for the detection of polycyclic aromatic hydrocarbon^.^ Since the epi~
(1) Cheng, Y. F.;Dovichi, N. J. Science 1988,242,562-564. (2) Zhu, H.; Clark, S. M.; Benson, S. C.; Rye, H. S.; Glazer, A N.; Mathies, R. A. Anal. Chem. 1994,66,1941-1948. (3) Clark, S. M.; Mathies, R. A. Anal. Biochem. 1993,215,163-170. (4) Nie, S.;Dadoo, R.; Zare, R N. Anal. Chem. 1993,65,3571-3575. (5) Huang. X.C.: Quesada, M. A; Mathies, R A Anal. Chem. 1992,64,21492154. (6) Huang, X. C.; Quesada, M. A; Mathies. R. A Anal. Chem. 1992,64,967972. (7) Nie. S.;Chiu, D. T.; Zare, R N. Science 1994,266. 1018-1021. (8) Taylor, J. A; Yeung, E. A Anal. Chem. 1993,65,956-960. (9) Wooley, A T.; Mathies. R. A Proc. Natl. Acad. Sci. (I.SA.1994,91, 1134811352. 0003-2700/95/0367-3367$9.00/0 0 1995 American Chemical Society
illumination confocal detection format allows the object freedom of movement throughout the focal plane, it is possible to adapt this detection geometry to scan the entire length of the capillary. The system we present is similar to the capillary array electrophoresis system described by Mathies and c o - w o r k e r ~for ~~~ continuously monitoring the outlet ends of several capillaries in a bundle. We have adapted Mathies's geometry to scan the entire length of a single capillary. In this paper we present the experimental details for a laser confocal fluorescence detector designed to scan the length of the separation capillary. The design of the laser scanner and present results on detection limits, minimizing separation time, and a method for measurement of solute diffusion coefficient independent of peak width contributions from the electrophoresis are reported. EXPERIMENTALSECTION Instrumentation. The confocal laser fluorescence detector is shown schematically in Figure 1. The output radiation (488 nm) from an air-cooled Omnichrome Model 532-5BS argon ion laser head passes through a neutral density filter (Omega Optical, Brattleboro, VT) and is elevated to -30 cm above the main breadboard (TMC, Inc., Atlanta, GA) surface with a Melles Griot (Imine, CA) precision beamsteering device. Using the tube current control on the laser head and various combinations of OD 1.0 and 0.3 neutral density filters, the laser power transmitted to the capillary can be controlled over a 0.6-25 mW range. The incident beam is reflected by a dichroic beamsplitter (Omega Optical) and focused into the capillary by a Zeiss 0.42 N.A., 32x Plan Achromat long working distance infinite conjugate microscope objective. The emission signal is collected by the same objective and transmitted back through the dichroic beamsplitter. A mirror above the dichroic beamsplitter reflects the emission beam through an interference filter centered at 530 nm (Omega Optical). The emission signal is focused by a fused silica focusing lens (Melles Griot) through a 200 pm pinhole (Melles Griot) and a long pass edge filter (515 nm, Omega Optical). The entire emission train is mounted on a Melles Griot 2 in. thick breadboard mounted directly to the main optical breadboard on legs 25.4 mm high (Melles Griot). The photon signal is converted to a current by a Hamamatsu (Bridgewater, NJ) 1P28 photomultiplier tube. The current is converted to a voltage, amplitied, and filtered by a Stanford Applied Research Systems low-noise SR750 current amplifier. Data are collected through a 12-bitA/D board (Keithley Metrabyte DAS 1602, Taunton, MA) and stored on an IBM PC clone using software written in-house. The capillary, 25-100 pm i.d. x 365 pm 0.d. (Polymicro Technologies, Phoenix, AZ), is mounted on an in-house constructed Plexiglas stage whose base and top were milled flat. A Analytical Chemistry, Vol. 67, No. 18, September 15, 1995 3367
Microscop Objective
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FIgure 1. Diagram of the laser confocal scanner.
0.3 mm groove was milled down the center of the top to receive the capillary and maintainalignment 'The sides of the stage were milled separately from the body of the stage and held in place with set screws to allow for 6ne adjustment of the capillary down the groove. A swagelock union is threaded into each side, concentric with the milled groove on the stage. Once the capillary is positioned in the groove, it can he locked into place with a nut and Vespel fermle swaged to the outer end of the union. 'This results in a bend in the capillary on the stage. 'The capillary is pulled tight in the groove by backing off - 4 4 turn on each union. Alignment of the beam down the length of the tube is achieved by fillingthe column with fluorescein and measuring the response at each end of the capillary. F i e adjustments of the entire stage on the positioning table are made until the response is equivalent at both ends of the capillary. The column is then scanned to ensure that the signal is uniform down the entire length of the column. ?he capillaries were stripped of the polyimide coating using either a butane tlame or fuming sulfuric acid and washed with water followed by methanol. We found that W-transparent coatings tended to v a in~thichess ~ Within a spool, which resulted in higher background noise levels relative to the stripped capillaries. If the polyimidestripped capillary is handled with reasonable care, breakage during installation is rare. With the current stage design, 19 an of a 26 cm long capillary can be scanned. 3368 Analytical Chemisty, Vol. 67, No. 18, September 15, 1995
The Plexiglas stage is mounted directly to a CP5W precision linear positioning table (Design Components, Inc., Franklin,MA), which is anchored to an optical breadboard. 'The positioning table is driven by a servo motor (Design Components, Inc.), controlled by an encoder device (Design Components, Inc.) connected directly to an IBM PC clone. 'The software allows the operator to program motion in a stepper motor configuration (20OOO steps/ in. resolution), with the encoder translating to an analog signal to the motor. The table can travel with a maximum velocity of 50 mm/s. Straight line a m r a c y and position location are better than 5 pm. In the scanning mode, the motor is triggered by a 5 V output from the data acquisition board to synchronize table motion with data collection. A Spellman (Planeview, NY) CZElooOR highvoltage power supply was used to generate the electrical field across the capillary. Columns were coated with a linear polyacrylamide according to the method described by Hjerten.'O 'The separation potential was always kept below 15 kV, and the current through the capillary did not exceed 45 pi% Injections were accomplished electroldn e t i d y . No provision was made for a load resistor parallel to the capillary to minimize rise and fall time of the injection voltage?' Reagents and chemicals. Fluorescein isothiocyanate 0 and all buffers were purchased from Aldrich (Milwaukee, W. (IO) Hjerteten. S.J. Ckmmntogr. 1985.347. 191-198 (11) Huang, X,Coleman. W.F.: &e. R N.J.Chmnotogr. 1989.480.95-110.
10
Figure 2. Plot of relative fluorescence response as a function of the laser power and dwell time per pixel.
Amino acids and proteins were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. FITClabeled insulin and lentil lectin were purchased from Sigma and used without further purification. Other proteins were labeled with FITC (limiting) in a 1:lmole ratio, in bicarbonate buffer, pH 9.5, and dialyzed against water to remove unreacted FITC. The protein was then diluted to the appropriate concentration in the operating buffer, as indicated in the text. Glycine was derivatized under the same conditions, with FITC as the limiting reagent. Acrylamide and TEMED were purchased from Bio-Rad labs (Melville, NY) and used without further puriiication.
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Figure 3. Capillary isoelectric focusing, without mobilization, of FITC-myoglobinin pH 3-10 ampholytes. An “ampholytes-only”scan has been background subtracted from the raw data.
RESULTS AND DISCUSSION In this paper, the fundamental considerations for characterization of the LIF scanner and preliminary data indicating performance are presented. One of the important experimental parameters to be investigated is detector response as a function of both laser power and dwell time per ~ i x e l . ~Optimization ~J~ of the signal entails balancing laser power with integration time over the pixel (dwell time per pixel) to extract as many photons as possible while minimizing photodestruction. The expected profiles should fit an exponential profile that accounts for laser intensity and the probability that a molecule will be intact after traversing the beam.I2 Based on the model for photon emission as a function of laser power and illumination time developed by Mathies and StryerI2 for photon counting, we tailored an experiment to our system for the optimization of laser power with dwell time per pixel (scan rate). In Figure 2 are shown the results of varying laser power over the range of 0.6-10 mW and dwell time for a 100 pm i.d. column. Two scans were made over the capillary for each data point. The first was made in a capillary filled with M fluorescein (signal). The second was made on a column filled with buffer solution (blank). The plotted response is signal readout voltage divided by the square root of blank readout voltage.12 At each power, 10 scan speeds were measured, and at each scan speed, 10 laser power levels were measured. This proved to be a sufficient number of points to reproduce the expected exponential profile. Dwell time per pixel is determined by the sampling rate, which was adjusted in concert with scan speed to keep a constant 20 pm/pixel width.
The curves follow the expected profile predicted by Mathies.12 Long dwell times and moderate laser power, 3-6 mW, are indicated to bum high onto the response curve for good S/N performance. Our data correlate well with Mathies’s theory in the high scattering background situation. Ideally, very long dwell times are indicated; however, to maintain reasonable analysis time, the dwell time was always less than 40 ms. We investigated similar response surfaces for different capillaries with inner diameters ranging from 25 to 100 pm. The shapes of the curves were all similar, but S/N decreased with smaller diameter capillaries. We attribute this to the difficulty in maintaining a good focal point through a thick capillary wall and distortions due to inhomogeneities in the capillary surface leading to distortions of the beam profile. In light of this, we currently work with 75 or 100 pm i.d. columns. The response curves also provide a guide for selecting optimum laser power for a given migration velocity or scan rate, or vice versa. Our initial objective in developing the laser scanner was to monitor, in situ, zones separated by capillary isoelectric focusing without mobilization. Pawliszyn and Wu14-16have reported the use of concentration gradient and absorbance imaging to detect focused zones without mobilization in short capillaries. We report here the detection, without mobilization, of fluorescently labeled proteins separated by isoelectric focusing. In Figure 3 is the isoelectropherogram, without mobilization, of FITC-myoglobin in pH 3-10 ampholytes with 10 mM sodium hydroxide as the catholyte and 20 mM phosphoric acid as the anolyte. The capillary was scanned at 5.08 mm/s using -6.5 mW laser power. Data were acquired at a rate of 254 Hz, to give a pixel width of 20 pm/ pixel. The background from a previously acquired “ampholytesonly” run was subtracted from the myoglobin isoelectropherogram to yield Figure 3. Several peaks can be observed in the isoelectropherogram; this is most likely due to the fluorescent label attached at different positions and multiply labeled molecules. The concentration of FITC-myoglobin is M, based on the FITC concentration. In the scanning mode, for focused peaks, detection limits are M (-2.4 fmol), while detection limits in the static mode (that is, with the detection point fixed) are M (-0.2 amol) for FITC-
(12) Mathies, R. A.; Peck, K.; Stryer, L. Anal. Chem. 1990,62, 1786-1791. (13) Art, J. J.; Goodman, M. B. In Methods in Cell Biologv;Matsumoto, B., Ed.; Academic Press, Inc.: New York. 1993; Vol. 38, pp 47-77.
(14) Wu, J.; Pawliszyn, J. Anal. Chim.Acta 1995,299, 337-342. (15) Wu, J.; Pawliszyn, J. J. Chromatogr. B 1994,657,327-332. (16) Wu, J.; Pawliszyn, J. Anal. Chem. 1994,66,867-873.
Analytical Chemistry, Vol. 67, No. 78, September 75, 7995
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Figure 4. (A) FITC-insulin, FITC-myoglobin, FITC-lentil lectin, and FITC-BSA separated in a 6% T linear acrylamide gel capillary. The detection point was fixed to give an effective column length of 14 cm. (B) Detection point was fixed 8 cm from the cathode. (C) The voltage was interrupted after 10 min, and the capillary was scanned from the anodic end.
labeled glycine. The discrepancy of the detection limits in the static and scanning modes probably arises from a combination of contributions. The myoglobin signal is diluted over several different reaction products, while glycine has only a single reactive site. Scattering from inhomogeneities of the capillary wall increases the scanning background. Variations in the wall thickness along the capillary length will distort the focal point and emission beam profile. Fluid vibrations within the capillary during the scan will increase the scanning background signal. Further design improvements will be aimed at minimizing these effects through the use of square capillariesI6 and a step-scan approach. In the step-scan mode, capillary motion is ceased during the measurement period to reduce the background signal. Scanning the entire length of the capillary gives one the power to observe the progress of the separation and also minimizes analysis time. In fact, it may not require the entire separation bed to achieve the desired resolution for the most dif6cult pair. Analysis time can be decreased by either positioning the detector to utilize as short an effective column length as possible while still retaining the requisite resolution1i or scanning the column during the course of the separation. Once the desired resolution of the most difficult pair is achieved, the capillary can be flushed and prepared for the next analysis. These concepts are illustrated in Figure 4, in which FITClabeled insulin, myoglobin, lentil lectin, and bovine serum albumin are separated by size in a 6% T linear acrylamide gel. In Figure 4a, the effective column length is 14 cm (fixed detection point). Thirty minutes is required for the last component to migrate past the detection point. The same zones are completely resolved in less than 17 min with use of a 8 cm long effective capillary, as shown in Figure 4b. The resolution of all four zones is sufficient
for quantitation yet requires about half the analysis time. Figure 4c depicts a capillary scan over the last 8 cm of the capillary of the same sample, at 5.08 mm/s (254 Hz sampling rate) after 10 min of applied voltage. The total analysis time was less than 11 min. The voltage was interrupted during the scan, so each zone moves through the detector window with the same velocity. The 11 min analysis time represents about a 3-fold decrease over the fixed detection point situation with use of a 14 cm long separation capillary. If, at this point, resolution between a particular pair of compounds is insuflicient, the voltage could be reapplied and more of the separation bed utilized. Molecular weight can be estimated from a plot of log MW vs migration distance. Yet another advantage to wholecolumn scanning is manifested in the capability to directly measure solute diffusion coefficient. The experiment is similar to the “stopped flow” experiment described by Walbraehl and Jorgenson18 for capillary electrcphoresis and originally developed by Knox and McLarenlg for GC. The column was positioned with the beam about midway down the length of the capillary. The test solute (FITC-glycine in this case) was injected at the cathodic end of a coated capillary in which electroosmosis was eliminated. When the glycine peak migrates past the detection beam, the voltage is interupted. From this initial run, electrophoretic mobility and temporal variance can be determined. Temporal variance can be converted to spatial variance by multiplying by the square of the migration velocity. During the time the voltage is off, the zone is scanned several times. Spatial variance is measured directly since the response as a function of position within the capillary is measured. During the voltage off period, the only mechanism for increasing variance is diffusion, which can be determined from the Einstein equation: c?
= 2Dt
Therefore, a plot of spatial variance vs time (when voltage is off) yields the diffusion coefficient from the slope. This diffusion coefficient is determined independently of any other peak variance contributions from electrophoresis. If desired, the detector can be repositioned at the anodic end of the capillary and the voltage reapplied to migrate the zone past the new detector p o ~ i t i o n . ~ ~ J ~ The y-intercept of the curve gives an indication of nondiffusional sources of band-broadening. We made diffusion coefficient measurements of FITC-glycine in three different buffers, 25 mM Tris, pH 8.5; 4% T linear acrylamide, and 6% T linear acrylamide at a field strength of 67 V/cm and 2 h diffusion time. The diffusion coefficients were determined from 0.5 times the slope of the linear plot (P = 0.999) of spatial variance vs time. The experimentally determined diffusion coefficients were as follows: free-solution, 6.7 x cm2/s, which agrees with previously reported diffusion coefficients of dansylated amino acids;” 4% T linear acrylamide, 5.1 x cm2/s; and 6% T linear acrylamide, 2.5 x cm2/s. It was important to operate at such a low field strength to eliminate any effects from Joule heating. The results from further investigations into capillary temperature and diffusion coefficient measurements of larger biopolymers will be the subject of another paper. (18) Walbroehl. Y.; Jorgenson, J. W. J. Microcolumn
(17) Clark. B. K.; Vo-Dinh, T.; Sepaniak. M. J. Anal. Chem. 1995.67,680-683.
3370 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
Sep. 1989.1. 41-45 (19) Knox. J. H.: McLaren, L. Anal. Chem. 1964,36, 1477-1482.
CONCLUSIONS We have developed a laser-induced fluorescence detector for capillary electrophoresis in which the entire capillary can be scanned during the course of the electrophoretic run. The detection geometry is based on an epi-illumination format with confocal optical detection geometry. The optimization of laser power and dwell time per pixel are presented. It is apparent that there is an optimum laser intensity depending on solute transit time in the probe volume. Therefore, the capability to tune laser power is an important characteristic not only in our application but also for laser fluorescence detection in general. We have demonstrated the capabilities of the scanning detector in light of isoelectric focusing and minimization of analysis time. We have further presented a method for directly determining the diffusion coefficient. Once the diffusion coefficient is obtained, then
expected peak widths can be calculated and compared with experimental peak widths. ACKNOWLEDGMENT We gratefully acknowledge support from the UAB Graduate School and Department of Chemistry for this work. We also acknowledge Jerry Sewell and the machine shop, operated by the Department of Physics, for construction of the capillary stage. Received for review March 0, 1995. Accepted June 16, 1995.8 AC950236Z @
Abstract published in Advance ACS Abstracts, August 1, 1935.
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