Anal. Chem. 1999, 71, 4173-4177
Microchip Flow Cytometry Using Electrokinetic Focusing David P. Schrum,† Christopher T. Culbertson, Stephen C. Jacobson, and J. Michael Ramsey*
Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 µm downstream from the intersection. Particles with diameters of 1 and 2 µm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios. Within the past decade, microfluidic structures created utilizing microfabrication technology have been employed in liquid-phase chemical analyses for a variety of analytical techniques. The majority of the published work has concentrated on using electrokinetically driven separation schemes to separate and detect analytes of interest. The separation modes demonstrated thus far include capillary electrophoresis,1-6 free-flow electrophoresis,7 open-channel electrochromatography (OCEC),8 micellar electrokinetic chromatography,9,10 OCEC with solvent programming,11 and capillary gel electrophoresis.12-14 Fluidic devices have also been fabricated which incorporate sample handling with electro† Present address: Department of Chemistry, University of Redlands, P.O. Box 3080, 1200 East Colton Ave., Redlands, CA 92373-0999. (1) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258. (2) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (3) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481-1488. (4) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (5) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (6) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113. (7) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 28582865. (8) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373. (9) Moore Jr., A. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184-4189. (10) Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 51655171. (11) Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. (12) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949-2953.
10.1021/ac990372u CCC: $18.00 Published on Web 08/07/1999
© 1999 American Chemical Society
kinetic separations and include sample derivatization using pre-15 and postcolumn reactors,16,17 DNA restriction digests,18 and PCR amplification.19, 20 To date, the manipulation of fluid flows on chips for counting and sorting particles or cells has received little attention. Optical flow cytometry is a common and well-established method for counting and sorting particles on a large scale, and a recent review highlights some of the standard types of analyses performed using this technique.21 The particles or cells that are analyzed using cytometry flow one at a time past an incident laser beam using hydrodynamic focusing. This focusing is created by a sheath fluid which encases the sample stream.22 Recently, microfluidic structures have been employed for flow cytometry, in which the hydrodynamically focused fluid streams were generated by syringe pumps.23 These devices have been used for the analysis of fluorescently labeled leukocytes.24 In addition to these hydrodynamic schemes, cells and particles can also be moved around on a chip using electric fields. For example, negative dielectrophoresis has been demonstrated on a microchip to sort and trap latex particles and trypsinized mouse cells using quadrupole and octopole cages.25 Electrokinetic transport has also been employed for the manipulation of cells on a microchip26 and to focus ions and fluids into a narrow stream.27 In this paper, we describe microfabricated instrumentation that incorporates electrokinetic focusing to spatially confine particles and monitor their spectral characteristics. Using mixtures of fluorescently labeled and unlabeled particles, particle counting is (13) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (14) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (15) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (16) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (17) Fluri, K.; Fitzpatrick, G.; Chiem, N.; Harrison, D. J. Anal. Chem. 1996, 68, 4285-4290. (18) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (19) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (20) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176. (21) Gilman-Sachs, A. Anal. Chem. 1994, 66, 700A-707A. (22) Shapiro, H. M. In Practical Flow Cytometry; Shapiro, H. M., Ed.; Wiley-Liss: New York, 1995. (23) Sobek, D.; Senturia, S. D.; Gray, M. I. Solid-State and Actuator Workshop, Hilton Head, SC, 1994; pp 260-263. (24) Blankenstein, G.; Larsen, U. D. Biosens. Bioelectron. 1998, 13, 427-438. (25) Fiedler, S.; Shirley, S. G.; Schnelle, T.; Fuhr, G. Anal. Chem. 1998, 70, 1909-1915. (26) Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568. (27) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3212-3217.
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Figure 2. Detection setup used for coincident laser light scattering and fluorescence measurements.
Figure 1. Schematic of the microchip used for flow cytometry with electrokinetic focusing.
demonstrated along with the ability to measure size distributions. In addition, the relationship of both particle throughput and counting efficiency with the sample to focusing field strength ratio is reported. EXPERIMENTAL SECTION Chemicals. The buffer used in these experiments was sodium tetraborate (EM Science, Gibbstown, NY; 10 mM, pH 9.2). Fluorescently labeled Fluoresbrite YG 2.0 µm microspheres (average diameter ) 1.88 ( 0.05 µm) and Fluoresbrite YG 1.0 µm microspheres (average diameter ) 0.972 ( 0.028 µm) were obtained from Polysciences, Inc. (Warrington, PA). Unlabeled Polybead carboxylate microspheres (average diameter ) 1.94 ( 0.09 µm) were also obtained from Polysciences. Fluorescently labeled FluoSpheres amine-modified YG microspheres (average diameter ) 0.93 ( 0.04 µm) were obtained from Molecular Probes, Inc. (Eugene, OR). Microchip Device. A schematic of the cytometry microchip is depicted in Figure 1. The glass microchips were fabricated inhouse using standard photolithographic and wet chemical etching techniques previously described.6 Three microchips were used for these experiments and were designated A, B, and C. The channels on microchip A were 50 µm wide and 22 µm deep, the channels on microchip B were 54 µm wide and 24 µm deep, and the channels on microchip C were 52 µm wide and 25 µm deep. All channel widths were measured at half-depth. The reservoirs were bonded to the channel ends with epoxy, and electrical contact with the solutions was achieved by placing platinum wires into each of the reservoirs. Microchip Operation. Three high-voltage power supplies (UltraVolt, Ronkonkoma, NY) were connected to the sample, focus-1, and focus-2 reservoirs and independently controlled through multifunction I/O cards (PCI-MIO-16XE-50; National 4174 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Instruments, Austin, TX) in a Macintosh PowerPC G3 computer (Apple Computer, Inc., Cupertino, CA) running LabVIEW software (National Instruments). The waste reservoir was grounded. Sample was continuously infused into the focusing chamber. The sample stream width was controlled by varying the potential applied to the sample reservoir relative to the focusing reservoirs. The relationship between the observed focused sample stream widths and the focusing electric field strengths applied was determined experimentally for each chip, as described previously.27 Detection. The detection setup for the latex particles is depicted in Figure 2. For coincidence measurements, lightscattering and laser-induced-fluorescence (LIF) signals were acquired simultaneously. The laser excitation source (Ar+, 488 nm, 10 mW; Innova 300 FReD, Coherent, Palo Alto, CA) was brought to a focus ∼50 µm downstream of the focusing chamber. The incident angle of excitation was ∼45°. The scatter and fluorescence signals were collected simultaneously from the chip using a 20× microscope objective (N.A. 0.4; Edmund Scientific Company, Barrington, NJ). The scatter and fluorescence signals were spatially filtered (200 µm × 200 µm square) and then split and sent to two different photomultiplier tubes (PMT; Model 77348; Oriel, Stratford, CT) using a dichroic mirror (515DRLP; Omega Optical, Inc., Brattleboro, VT). The fluorescence signal was again spectrally filtered using a holographic notch filter (488 nm; Kaiser Optical Systems, Inc., Ann Arbor, MI) to reduce laser source noise. The signals from the PMTs were then amplified (428-MAN; Keithley, Cleveland, OH) and digitized with the same multifunction I/O cards and computer used for the voltage control. CCD fluorescence imaging experiments using the 0.93 µm amine modified fluorescently labeled particles and the 1.88 µm labeled particles were performed to study the confinement of the particles in the focusing region of the chip. An inverted microscope equipped with an epifluorescence attachment (Model TE300; Nikon USA, New York, NY) and CCD camera (Princeton Instruments, Inc., Trenton, N.J) was used to acquire the images. The acquired images were processed using IP Lab Spectrum software (Signal Analytics Co., Vienna, VA).
Figure 3. Time-integrated CCD image of electrokinetically focused 1.88 µm labeled particles on microchip A. The exposure time was 5 s with sample and focusing field strengths of 100 and 300 V/cm, respectively. Arrows depict direction of transport, and their lengths are proportional to average fluid velocities in each channel.
RESULTS AND DISCUSSION A 5 s integrated CCD exposure of particle transport through the focusing chamber is shown in Figure 3. The streaks observed in the microchip channels are due to the fluorescence from individual particles as they travel from the sample channel, through the focusing chamber, and down the analysis channel. The fluorescently labeled particles were 1.88 µm in diameter and at a concentration of ∼1.5 × 107 particles/mL. The lengths of the arrows correspond to the observed fluid velocities and are proportional to the field strengths in each of the channels. The stream width, measured at a point 50 µm beyond the focusing chamber, was 8.0 µm at maximum extent. This is ∼15% of the channel width. The sample to analysis field strength ratio was 0.15 (100 V/cm in the sample channel and 700 V/cm in the analysis channel). Figure 4 shows typical scatter/fluorescence coincidence data collected from a 50/50 mixture of 0.972 µm fluorescently labeled and 1.94 µm unlabeled latex particles. Data from such runs were generally acquired for 60 s to gather enough information for statistical treatment; however, only a 10 s segment of the run is shown so that the individual peaks can be seen. The inset in Figure 4 is an expanded view of a 0.4 s section of the run. Each peak in the scatter channel (A) represents a single particle passing through the interrogation region, with the labeled particles also appearing in the fluorescence channel (B). The sample to focusing field strength ratio (Esample/Efocus) was 0.18 and yielded a sample stream width of ∼7-8 µm. The average particle throughput was 13.5 particles/s, which is within experimental error of the expected 15 ( 1 particles/s. The expected particle throughput was calculated from the initial particle concentrations (3.3 × 107 particles/mL for each particle type), the particle electrokinetic mobilities, the cross-sectional area of the channel, and the sample channel field strength (110 V/cm). The observed mobilities of
Figure 4. (A) Light scattering and (B) fluorescence signals for a mixture of 0.972 µm labeled particles (3.3 × 107 particles/mL) and 1.94 µm unlabeled particles (3.3 × 107 particles/mL) collected on microchip B. The inset is an expanded view of a 0.4 s section of the run. The sample channel field strength was 110 V/cm, and the average focusing channel field strength was 600 V/cm. Table 1. Light Scattering Peak Intensity, Velocity, and Particle Size Distribution Data Obtained for Labeled and Unlabeled Particles light scattering peak intensity (arb) jx
σ2
σ2/xj2
particle velocity (cm/s) jx
σ2
σ2/xj2
particle size σ2/xj2
labeled 0.0869 0.000471 0.0624 0.0931 0.000520 0.0601 0.0023 unlabeled 0.371 0.00285 0.0207 0.0985 0.000157 0.0162 0.0045
the particles, 1.56 × 10-4 cm2/(V‚s) for the labeled particles and 1.65 × 10-4 cm2/(V‚s) for the unlabeled particles, were determined in separate single-point experiments. Higher sample throughputs can be obtained by increasing Esample/Efocus, but the sample and focusing field strengths applied to the microchip in this experiment were selected to give a narrow sample stream width, so that all particles would pass through the detector probe region. Baseline resolution is observed between each of the peaks in the scatter and fluorescence channels, as shown in the inset in Figure 4. The fluorescence signal (B) from the smaller (0.972 µm) particles in Figure 4 correlates well with the lower intensity peaks in the light scattering data (A). The corresponding peak intensity in the scatter channel for the 0.972 µm diameter labeled particles is 0.235 of the signal from the 1.94 µm unlabeled particles (Table 1). These observations are consistent with that expected for forward angle Mie scattering where the scattering intensity should be proportional to the particle surface area and, therefore, to the square of the particle diameter.28 (28) Bohren, C. F.; Huffman, D. R. Absorption and scattering of light by small particles; John Wiley and Sons: New York, 1983; pp 101-119.
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Table 2. Effect of the Esample/Efocus Ratio on Observed Sample Stream Width, Particle Throughput, and Detection efficiency
Esample/ Efocus
sample stream widtha (µm)
observed throughput (particles/s)b
expected throughput (particles/s)c
detection efficiency (% detected)
0.15 0.29 0.49 0.72
7.3 10.5 12.6 16.3
14.7 ( 0.6 22.0 ( 0.8 32.2 ( 1.4 34.0 ( 2.0
15 28 44 60
98 ( 4 79 ( 3 73 ( 3 57 ( 3
a Data generated on microchip C using 5 s integrated CCD images of labeled 0.93 µm particles (4.5 × 108 particles/mL). b Sample was a 1/1 mixture of labeled 0.93 µm and 1.94 µm unlabeled particles with a total particle concentration of ∼6.6 × 107 total particles/mL. c Calculated as described in text.
Figure 5. (A) Light scattering and fluorescent scatter plot and (B) light scattering histogram for the data shown in Figure 4. The boxes in (A) contain 95% of all the detection events. The arrow drawn in (B) separates the particle distribution based on the number of fluorescence events observed.
Figure 5A shows a scatter plot of the same set of data displayed in Figure 4. The boxed regions in Figure 5A contain 95% of the total number of particles that were detected in this experiment. Figure 5B shows a histogram of the peak intensities from the scatter data in Figure 5A. The histogram has a bimodal distribution, which is consistent with the two sizes of particles analyzed. The location of the arrow drawn in the plot was determined using the fluorescence channel data where 368 fluorescently labeled 0.972 µm particles were observed. This number was subtracted from the total number of peaks counted in the scatter channel (808) to obtain the number of peaks due to the unlabeled 1.94 µm particles (440). The two types of particles were also run individually to confirm that the two peaks seen in the signal intensity distribution in Figure 5B were actually due to the two different sized particles. To compare the observed ratio of labeled to unlabeled particles (368/440 or 0.836/1) with that expected (1/1), a correction for the mobility differences between the two types of particles (reported above) must be made, for the unlabeled particles have a mobility 1.06 times greater than the labeled particles. The corrected ratio of labeled to unlabeled particles is 0.887/1 which is within experimental error of the 1/1 mixture that was used. The observed experimental distributions in peak intensities seen in Figure 5B are a convolution of two independent distributions: a particle size distribution and a particle velocity distribution. The variance in the light scattering intensity distribution (σ2intensity;Table 1), therefore, is the sum of the variances for the particle size (σ2particle) and velocity (σ2velocity) distributions. The velocity distributions were determined experimentally in separate, single-point-detection experiments. In these experiments gated injections16 of the particles were made at field strengths similar 4176 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
to those used in the cytometry experiments (Figure 4). From the variation in the individual particle velocities, the velocity distributions were calculated and are given in Table 1. The variances (σ2) were normalized by dividing each variance by the square of its mean (σ2/xj2). These normalized variances show that the distribution in particle velocities is responsible for 96% and 78% of the total peak intensity distribution for the labeled and unlabeled particles, respectively. To compare the particle size distribution determined from the cytometry measurements with those given by the manufacturer, the relative standard deviation (RSD) for the observed cytometry data is divided by 2 because the intensity data is proportional to the square of the diameter. Using this approach, the RSDs of the particle size distribution for the 0.972 µm labeled particles and for the 1.94 µm unlabeled particles were determined to be 2.4% and 3.3%, respectively. These values are close to the RSD values of 2.9% and 5% reported by the manufacturer. As mentioned above, a tradeoff exists between sample stream width and the sample throughput. The sample stream width, and consequently the sample throughput (Table 2), is a function of the sample to focusing field strength ratio (Esample/Efocus). All of the sample stream widths reported in Table 2 were determined using integrated CCD images (similar to Figure 3) of the 0.93 µm fluorescently labeled particles and are reported at their full width. The expected particle throughput was determined using the initial sample concentration, the sample channel field strength, the observed particle mobilities, and the cross-sectional area of the sample channel. The observed mobilities of the particles were 2.54 × 10-4 cm2/(V‚s) for the 0.93 µm labeled particles and 1.65 × 10-4 cm2/(V‚s) for the 1.94 µm unlabeled particles. When the stream width was 7.3 µm the particle throughput is low, but all of the particles are expected to flow through the probe region, and within experimental error they do. To increase the particle throughput, the sample to focusing field strength ratio is increased. When this is done, however, the stream width increases and the detection efficiency decreases when the probe volume remains constant (see Table 2). For these experiments the spatial filter defined a probe area of 10 µm × 10 µm square. The widest stream width generated in these experiments was 16.3 µm, which was approximately 1.6 times wider than the detector probe region. Under these conditions we estimate that only 57% of the particles were detected. If higher throughputs are needed, one option to
Table 3. Particle Mixture Data Obtained with Labeled and Unlabeled Particles on Microchip C concentration mixture ratioa Esample/ unlabeled/labeled unlabeled/labeled (expected)b (experimental)c (unlabeled/labeled) Efocus 23/1 23/1 23/1 10/1 10/1 10/1 1/1 1/1 1/1
0.15 0.29 0.49 0.15 0.29 0.49 0.15 0.29 0.49
15 ( 1 15 ( 1 15 ( 1 6.5 ( 0.1 6.5 ( 0.1 6.5 ( 0.1 0.65 ( 0.01 0.65 ( 0.01 0.65 ( 0.01
16 ( 2 15 ( 1 15 ( 3 7(3 7(2 6(1 0.69 ( 0.03 0.67 ( 0.03 0.72 ( 0.08
a Samples consisted of labeled 0.93 µm particles and 1.94 µm unlabeled particles. b Unlabeled/labeled ratio expected after accounting for the mobility differences between the two types of particles. c Average unlabeled/labeled ratio collected from four 60-s runs.
improve the detection efficiency is to increase the width of the probe region. This, however, increases the probability of having multiple particles in the detection window simultaneously. Sample mixtures consisting of 0.93 µm amine-modified labeled particles and 1.94 µm unlabeled particles were also analyzed, with the results shown in Table 3. The samples were mixed in 1/1, 10/1, and 23/1 ratios of unlabeled/labeled particles. The total particle concentration for each ratio was ∼6.6 × 107 particles/ mL. The expected ratios were corrected for the mobility differences between the two types of particles as shown in Table 3. In this case, the amine labeled particles had a mobility 1.54 times greater than the unlabeled particles. All of the experimentally observed particle ratios are within experimental error of their respective expected ratio. As the data in Table 3 indicate, it is possible to distinguish between varying mixtures of fluorescently (29) Wolff, A.; Larson, U. D.; Blankenstein; Philip, J.; Telleman, P. Proceedings of the µTAS’98 Workshop, Banff, Alberta, Canada, 1998; pp 77-88.
labeled and unlabeled particles, based upon the measured scattering and fluorescence profiles obtained. In conclusion, microchip cytometry with electrokinetic focusing was performed to spatially confine samples of particles varying in type, size, and ratio, and to count these particles using coincident light scattering and fluorescence detection. Particles of varying sizes can be distinguished by their scattering intensities. In addition, sample throughput and detection efficiency were shown to be affected by the sample to focusing field strength ratio applied to the chip. Microchip cytometry has the potential to be less expensive, consume less solvent, and require less bulky instrumentation than conventional flow cytometry. Rare event cell sorting can also be carried out on a microchip, without the dilution which occurs with conventional flow cytometers.29 Methods of increasing sample throughput, such as applying higher operating field strengths and using microchips with different and/or multiple channel geometries, are being explored to achieve 0.1-1 kHz counting rates. ACKNOWLEDGMENT This research was sponsored by the Department of Energy Office of Research and Development. Oak Ridge National Laboratory (ORNL) is managed by Lockheed Martin Energy Research Corporation for the U.S. Department of Energy under Contract DE-AC05-96OR22464. This research was supported in part by an appointment for D.P.S. to the ORNL Postdoctoral Research Associates Program, administered jointly by ORNL and the Oak Ridge Institute for Science and Education. The authors thank Christopher D. Thomas and John W. Cockfield for preparation of the microchips.
Received for review April 8, 1999. Accepted June 24, 1999. AC990372U
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