`
`Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System
`on a Chip
`Author(s): D. Jed Harrison, Karl Fluri, Kurt Seiler, Zhonghui Fan, Carlo S. Effenhauser and
`Andreas Manz
`Source: Science, New Series, Vol. 261, No. 5123 (Aug. 13, 1993), pp. 895-897
`Published by: American Association for the Advancement of Science
`Stable URL: https://www.jstor.org/stable/2882118
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`Agilent Exhibit 1273
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`

`

` Micromachining a Miniaturized Capillary
` Electrophoresis-Based Chemical Analysis
` System on a Chip
`
` D. Jed Harrison,* Karl Fluri, Kurt Seiler, Zhonghui Fan,
` Carlo S. Effenhauser, Andreas Manz
`
` REPORTS
`
` berta Microelectronic Centre (AMC) (Ed-
` monton, Canada). Devices made at AMC
` used Corning 7740 Pyrex, polished and
` then fine-annealed. A solution of HF:
` HNO3 [200 ml (49% HF) and 140 ml (70%
` HNO3) diluted to 1 liter] was used to etch
` the glass to a depth of 10 to 20 ,um through
` a Au and Cr metal mask. An electron
` micrograph of an intersection between two
` Micromachining technology was used to prepare chemical analysis systems on glass chips (1
` channels is shown in Fig. 1. The character-
` centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and
` istic curvature of the walls associated with
` electrophoretic separation to distinguish sample components. Capillaries 1 to 10 centimeters
` isotropic etching as well as the relative
` long etched in the glass (cross section, 10 micrometers by 30 micrometers) allow for capillary
` smoothness are clearly apparent. With a
` electrophoresis-based separations of amino acids with up to 75,000 theoretical plates in about
` photomask feature width of 10 jim and an
` 15 seconds, and separations of about 600 plates can be effected within 4 seconds. Sample
` etch depth of 10 ,um, the channels were
` treatment steps within a manifold of intersecting capillaries were demonstrated for a simple
` about 30 ,um wide at the surface. The yield
` sample dilution process. Manipulation of the applied voltages controlled the directions of fluid
` of devices was about 70% and was appar-
` flow within the manifold. The principles demonstrated in this study can be used to develop a
` ently limited by defects in the glass. A top
` miniaturized system for sample handling and separation with no moving parts.
` plate of Coming 7740 glass with access
` holes drilled into it was thermally bonded
` to the etched plate at 6500 to 660?C for 4 to
` 6 hours. This process was repeated two to
` Photolithography and chemical etching
` glass substrate (13, 14). In this report we
` three times to eliminate bonding defects
` techniques have been combined to create
` show that very rapid separations (<5 s) of
` (14). The glass devices prepared at the
` the field of micromachining (1, 2), in
` fluorescently labeled amino acid mixtures
` commercial facility were bonded at 6200C
` which three-dimensional microstructures
` can be effected within such devices. Fur-
` for 4 hours. The remaining figures illustrate
` such as motors, tweezers, beams, pumps,
` thermore, we were able to control the flow
` various channel layouts that were fabricat-
` and valves have been fabricated on the
` rate and flow direction of the solvent within
` ed. Plastic pipette tips inserted in the holes
` micrometer scale (3). This technology has
` a manifold of intersecting channels by ap-
` in the cover plate acted as reservoirs into
` attracted considerable interest in the devel-
` plying voltages to each of the capillary
` which buffer and Pt electrodes were placed.
` opment of both sensors and actuators, and
` channels simultaneously (5, 14). Both sam-
` The rapid separation of six amino acids
` sensors for physical forces are becoming
` ple dilution and injection can be effected
` labeled with y-fluorescein isothiocyanate
` well developed (1). However, the use of
` with this technique, as described below.
` (FITC), along with the hydrolysis products
` micromachining to fabricate chemical sen-
` Taken in combination, these results show
` of the fluorescent label, is shown in Fig. 2.
` sors, chemical analysis systems, or even
` that it will be possible to develop a com-
` The individual 1 mM amino acids were
` laboratories on the scale of a silicon chip
` plete, miniaturized, integrated system with
` allowed to react with 0.1 mM FITC in pH
` remains in its infancy, despite the consid-
` sample pretreatment, separation, and de-
` erable promise this approach appears to
` tection on a "chip." The use of electroos-
` offer (4-9). Compared to conventional sys-
` motic pumping offers an additional advan-
` tems, such devices could reduce solvent and
` tage compared to micromachined pumps
` sample consumption or decrease analysis
` and valves (1) in that there need be no
` times because of their decreased dimensions
` moving parts, and reliable performance can
` (4, 5). Integration of several different pro-
` be achieved with present technology.
` cesses on a single chip to form a system for
` We fabricated devices by using standard
` sample pretreatment, separation, and de-
` microphotolithographic techniques (1, 2),
` tection may allow for new overall processes,
` either at a commercial facility (Baumer
` improve efficiency, make possible automa-
` IMT, Zurich, Switzerland) or at the Al-
` tion, and reduce manufacturing costs (5).
` Electrokinetic effects could be used to
` advantage in a miniaturized analysis system
` (5). Electroosmotic flow provides a pump-
` ing method that is convenient for small
` capillaries that develop high back pressures
` with conventional pumps, and electropho-
` retic separation has been shown to be an
` extremely useful chemical separation tech-
` nique (10-12). We have recently shown
` that capillary electrophoresis (CE) can be
` performed in capillary channels etched in a
`
` Buffer Sample
`
` 1 2L
`
` 10 FM Amino acids Waste
` pH 9.0 Waste Detector
`
` (1.06 kV/cm)
` did =2.2 cm
`
` 3 4
`
` 657
`
` 0 4 8 12 16 20
` Time (s)
`
` Fig. 2. Electropherogram of six FITC-labeled
` amino acids in pH 9.0 buffer with 2330 V
` applied between the injection and detection
` points and a potential applied to the side chan-
` nels to reduce leakage of the sample. The
` peaks were identified by the separate injection
` of each component and are as follows: 1, Arg;
` 2, FITC hydrolysis product; 3, Gln; 4, Phe; 5,
` Asn; 6, Ser; and 7, Gly. The inset shows the
` approximate layout of the device, with a buffer
` to separation channel-waste distance of 10.6
` cm.
`
` D. J. Harrison, K. Fluri, K. Seiler, Z. Fan, Department of
` Chemistry, University of Alberta, Edmonton, Alberta,
` Canada T6G 2G2.
` C. S. Effenhauser and A. Manz, Corporate Analytical
` Research, Ciba-Geigy Ltd., CH4002 Basel, Switzer-
` land.
`
` *To whom correspondence should be addressed.
`
` Fig. 1. Electron micrograph of capillary chan-
` nels etched into Corning 7740 glass to a depth
` of 1 0 ,um.
`
` SCIENCE * VOL. 261 * 13 AUGUST 1993 895
`
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`Agilent Exhibit 1273
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`
`

`

` while positioned on an Olympus BH-2 micro-
` glass structure is capable of resolving many
` 9 buffer (20 mM boric acid and 100 mM
` scope stage (20:1 magnification). The applied
` components of a complex sample mixture
` tris) for 16 hours. These samples were
` potential drove buffer along the horizontal
` with an efficiency equal to that of conven-
` diluted to 10 ,uM in formal concentration
` channel, while the channel running vertically
` tional CE for a similar applied potential.
` of the amino acids. Excitation of the fluo-
` in Fig. 4 was filled with fluorescent dye and
` A very rapid separation within a smaller
` rescent labels was effected with the 488-nm
` left floating. There is convective flow of the
` device with a separation distance of 0.75
` line of an Ar ion laser (14-1 7). For the data
` dye out of the side channel, and the dye mixes
` cm is illustrated in Fig. 3. Three amino
` in Fig. 2, we injected a sample plug by
` acids of differing charge were separated
` applying 2 kV (1.2 kV/cm) for 10 s between
` with the buffer downstream of the intersection
` point. The magnitude of the effect is more
` within 3 s, with 2.5 kV applied (1.56
` the sample reservoir and the waste reservoir
` pronounced at high applied potentials, and
` kV/cm), corresponding to 1.17 kV across
` shown at the bottom of the inset in Fig. 2.
` mixing occurs over an extended distance
` We then separated the plug while applying
` the separation region. A 1-s injection time
` downstream. It is significant that the leakage
` was used with 500 V applied between sam-
` 11.25 kV (1.06 kV/cm) between the buffer
` arises from a convective effect and is not due
` ple and injection-waste reservoirs. The sep-
` reservoir and the waste reservoir on the
` to diffusion alone, as it indicates that the
` right of the inset in Fig. 2; the total analysis
` aration efficiency corresponded to about
` resistance to flow of side channels may be
` 600 plates but may be improved if the
` time was 25 s. The injection to detection
` manipulated to reduce leakage.
` injection parameters are optimized. This
` distance was 2.2 cm, corresponding to 2.3
` In principle, convective flow at inter-
` rapid cycle time illustrates that the response
` kV across the region of separation, and the
` secting channels could be controlled by
` time of such a device can compete with the
` number of theoretical plates obtained
` controlling the potential of all reservoirs
` response time of many chemical sensors.
` ranged from 40,000 to 75,000 for the dif-
` that contact the intersection. The result of
` Furthermore, rapid separations are required
` ferent amino acids. This corresponds to 17
` such an experiment for a T intersection is
` if CE is to be considered for the second
` to 32 plates per volt, which is equivalent to
` shown in Fig. 5. The applied potentials
` stage of a two-dimensional separation,
` results in conventional fused silica capillar-
` caused flow from the buffer and fluorescein
` where CE is combined with a technique
` ies (15). The results show that the planar
` sample reservoirs toward the waste reser-
` such as liquid chromatography (LC) as the
` voir. These two solutions mixed down-
` first stage (18). Integration of both CE and
` stream of the T intersection point as they
` LC manifolds on a single chip provides an
` flowed toward the detector, diluting the
` attractive means of circumventing problems
` fluorescein dye. An increase in the poten-
` with dead volumes. This work shows that a
` tial of the buffer reservoir increased the flow
` CE system on a chip is indeed feasible.
` of buffer and further diluted the sample
` Fabrication of an LC system in Si has been
` solution downstream of the intersection.
` reported (19).
` The fluorescence intensity decreased as the
` We have been able to use this system to
` potential of the buffer channel was in-
` perform quantitative analysis of the amino
` creased as a function of time, illustrating
` acids by varying the size of the injection
` the dilution effect. This result also shows
` plugs or the sample concentration. The
` that control of the potential of all of the
` labeled amino acids were prepared by reac-
` channels could be used to control the leak-
` tion of 1.7 mM FITC with 8.3 mM amino
` age phenomenon. More importantly, a
` acids in pH 9.1 carbonate buffer (60 mM
` carbonate) with 0.001% pyridine for 16
` hours and were then diluted with pH 9.1
` buffer. The peak areas varied linearly with
` labeled Arg and Tyr concentrations be-
` tween 0.05 and 20 ,uM (assuming complete
` /# L....... -3 kV
` reaction with FITC). A precision of ? 2 to
` / waste
` 2:1 Detector
` 4% was obtained for both amino acids,
` based on the error in slope and intercept of
` the calibration curve. Similarly, increasing
` the sample plug size at constant concentra-
` tion from 200 to 1800 ,m gave a linear
` increase in peak area, with +?2% precision
` 1 0-
` on the basis of the error in slope of a plot of
` peak area versus plug length.
` When the potential of a side channel is
` left uncontrolled, solution may leak into
` the active channel. For the device used in
` Fig. 3, control of the potential at only the
` buffer and waste reservoirs during separa-
` tion led to -3 to 4% leakage from the
` sample channel. The increase in the back-
` Fig. 5. Fluorescence intensity downstream of
` ground fluorescence signal this causes de-
` the T intersection is shown as a function of time,
` pends on the layout of the device and can
` while the potential on the buffer channel was
` reach 20 to 30%.
` increased with time. The flow of buffer dilutes
` A photomicrograph of a device taken
` the 10 F~M fluorescein dye coming from the
` while two of the three channels were actively
` sample channel; the extent of dilution is con-
` under potential control is shown in Fig. 4.
` trolled by the potential of the buffer channel
` The device was flooded with 488-nm light
` relative to the sample channel.
`
` Conditions:
` 50 kM Amino acids
` pH 8.0 Tris/boric acid
` 2500 V (1 562 V/cm)
` dId = 0.75 cm
`
` 2
`
` Waste (for injection)
`
` Buffer ___
`
` Waste
` (for
` separation)
`
` I I I I Sample
` 0 2 4 6 8
` Time (s)
`
` Fig. 3. Electropherogram of 10 ,uM FITC-la-
` beled Arg, Phe, and Glu (peaks 1, 2, and 3,
` respectively) in pH 8.0 buffer in a device 1 cm
` by 2 cm, with an injection to detection distance
` of 0.75 cm.
`
` Sample Bufferchannel
` GND r Flow channel
`
` -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~P.--
`
` ?400-
`
` Fig. 4. Photomicrograph of a device with 10 kV
` (1 kV/cm) applied along the horizontal channel,
` while the side channel (vertical in the photo)
` was left floating. The potential drove buffer
` solution (pH 9.1) from left to right and caused a
` 100 ,uM fluorescein dye solution in the side
` channel (vertical in the figure) to be pulled in
` the direction of flow. The channels are 30 p.m
` wide.
`
` m 0 2 4 6
` Time (min)
`
` 896 SCIENCE * VOL. 261 * 13 AUGUST 1993
`
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`
`Agilent Exhibit 1273
`Page 3 of 4
`
`

`

` MSREPORTS
`
` 18. C. A. Monnig and J. W. Jorgenson, Anal. Chem.
` 63, 802 (1991).
` 19. A. Manz et al., Sens. Actuators B 1, 249 (1990).
` 20. We thank G. McKinnon and G. Fitzpatrick for
` valuable assistance and both the Natural Scienc-
`
` es and Engineering Research Council of Canada
` and Ciba-Geigy for support. Z.F. thanks AMC for
` a research stipend.
`
` 10 March 1993; accepted 8 June 1993
`
` High-Density Nanosecond Charge Trapping in Thin
` Films of the Photoconductor ZnODEP
`
` Chong-yang Liu, Horng-long Pan, Marye Anne Fox,*
` Allen J. Bard*
`
` REFERENCES AND NOTES
`
` common sample pretreatment step, dilu-
` tion, can be effected within the capillary
` channel manifold. Typical sample prepara-
` tion steps performed within a conventional
` flow injection analysis system could also be
` effected within these devices by electroos-
` motic pumping of the fluid phase.
` Taken together, our results show that
` the micromachining of capillary channels
` on planar substrates provides a route to
` fabricating miniaturized chemical systems
` on a chip capable of quantitative analysis.
` Rapid separation of complex sample mix-
` An electrooptical memory effect is observed with solid thin films of the photoconductor
` tures combined with sample handling steps
` zinc-octakis(,-decoxyethyl) porphyrin (ZnODEP) sandwiched between two optically trans-
` such as dilution and injection provide a
` parent electrodes. Upon irradiation with the simultaneous application of an electric field,
` basis for more complex, miniaturized anal-
` electron-hole pairs are generated and separated within the photoconductive layer. These
` ysis systems. The application of microma-
` electron-hole pairs become "frozen" within the films when the irradiation is interrupted.
` chining techniques to the miniaturization
` These trapped charges can be released by irradiation of the cell, resulting in a transient
` of chemical analysis is very promising and
` short-circuit photocurrent. No cross talk between adjacent memory elements separated by
` should lead to the development of analyti-
` -0.2 micrometer (a density of 3 gigabits per square centimeter) was detected. The charge
` cal laboratories on a chip.
` storage system is robust and nonvolatile. The response time for the write-read beam is in
` the subnanosecond range, and no refreshing is required for long-term retention of trapped
` charges.
`
` 1. K. D. Wise and K. Najafi, Science 254, 1335
` (1991).
` 2. K. E. Petersen, Proc. IEEE70, 420 (1982).
` 3. L. S. Tavrow, S. F. Bart, J. H. Lang, Sens. Actua-
` torsA 35, 33 (1992); C.-J. Kim, A. P. Pisano, R. S.
` electrons into the irradiated electrode.
` A nonvolatile, rewritable electrooptical
` Muller, M. G. Lim, in Technical Digest, IEEE 1990
` While investigating the behavior of such
` method of data storage has been developed
` Solid State Sensor andActuator Workshop, Hilton
` cells under a constant bias with pulsed
` that is based on charge trapping in thin
` Head Island, SC, 4 to 7 June 1990 (IEEE, New
` York, 1990), pp. 48-51; M. Esashi, S. Shoji, A.
` irradiation or with a pulsed bias under
` films of a photoconductive material, in this
` Nakano, Sens. Actuators 20, 153 (1989); S. F.
` steady illumination, we observed charge
` case, ZnODEP, with which high densities
` Bart, M. Mehregany, L. S. Tavrow, J. H. Lang,
` trapping.
` and high speeds can be achieved. This
` Sens. Actuators A 21-23, 193 (1990).
` 4. A. Manz, N. Graber, H. M. Widmer, Sens. Actua-
` Cells (Fig. 2) were constructed as previ-
` information storage method requires a ma-
` tors B 1, 244 (1990).
` ously described (5) by capillary filling of
` terial with a high resistance in the absence
` 5. A. Manz et al., Trends Anal. Chem. 10, 144 (1991).
` molten ZnODEP (6, 7) into the 1- to 2-,um
` of light, good photoconductivity, and the
` 6. S. C. Terry, J. H. Jerman, J. B. Angell, IEEE Trans.
` gap between two ITO electrodes (area, 0.5
` Electron Devices ED-26, 1880 (1979).
` capacity to inject stored charge upon simul-
` 7. W. Olthus, B. H. van der Schoot, P. Bergveld,
` cm2). Upon solidification, the film was
` taneous application of light and an electric
` Sens. Actuators 17, 279 (1989).
` illuminated with a write beam (wavelength,
` field. Information, as trapped charge, can
` 8. S. Shoji, M. Esashi, T. Matsuo, ibid. 14, 101
` 550 nm) to produce a cathodic current
` be written, read, and erased by simultane-
` (1988); H. Suzuki, A. Sugama, N. Kojima, Sens.
` Actuators B 10, 91 (1993).
` when a negative potential was applied to
` ous application of an electric field and a
` 9. S. J. Pace, U.S. Patent 4,908,112 (1990).
` the irradiated ITO electrode (we choose the
` light pulse. We also used a scanning tun-
` 10. P. D. Grosman and J. C. Colburn, Eds., Capillary
` sign of the applied potential to be that of
` neling microscope (STM) for charge stor-
` Electrophoresis: Theory and Practice (Academic
` the irradiated, or front, electrode with re-
` Press, San Diego, CA, 1992).
` age (writing) and charge measurement
` 11. J. W. Jorgenson and K. D. Lukacs, Anal. Chem.
` spect to the back electrode). In this writing
` (reading) within elements as small as 40 nm
` 53,1298 (1981).
` step, initially vacant traps within the film
` in diameter.
` 12. A. G. Ewing, R. A. Wallingford, T. M. Olefirowicz,
` are filled with electrons (Fig. 3). Because
` Photoconductivity has been widely in-
` ibid. 61, 292A (1989).
` 13. A. Manz et al., J. Chromatogr. 593, 253 (1992).
` the resistivity of ZnODEP in the dark is
` vestigated (1), and the displacement of
` 14. D. J. Harrison, A. Manz, Z. Fan, H. Ludi, H. M.
` very high (>1014 ohmcm), electron move-
` charge in photoelectrets upon application
` Widmer, Anal. Chem. 64, 1926 (1992); K. Seiler,
` ment "freezes" when the light is switched
` of an electric field and light, a phenomenon
` D. J. Harrison, A. Manz, ibid. 65, 1481 (1993).
` 15. Y.-F. Cheng and N. J. Dovichi, Science 242, 562
` off; the trapped charge remains stored in the
` called persistent internal polarization, has
` (1988).
` been known for more than 35 years (2, 3).
` 16. D. J. Harrison, P. G. Glavina, A. Manz, Sens.
` Analogous effects are also important in
` Actuators B 10, 107 (1993).
` electrophotography (4).
` 17. The Uniphase/Cyonics laser was operated at an
` output power of 4 to 5 mW (15). Either the beam
` Our interest in this approach grew from
` was directed onto the sample through a 600-pm
` our investigations of solid-state photocells
` optical fiber or mirrors and a lens were used to
` composed of a solid film of the liquid crystal
` focus the beam to about 40 pm. Emission was
` collected with a 10:1 or 25:1 microscope objec-
` porphyrin ZnODEP (Fig. 1) held between
` tive and then directed into a photomultiplier tube
` transparent indium-tin-oxide (ITO) elec-
` after filtering with an Omega 508- to 533-nm
` trodes, first described by Gregg et al. (5).
` bandpass filter. The signal was electronically fil-
` tered and digitally collected with a Macintosh
` Such cells produce steady-state short-circuit
` computer equipped with a National Instruments
` photocurrents by preferential injection of
` NB-MIO-16 analog-to-digital converter and Lab-
` view software or an 8-bit digital LeCroy 9310
` oscilloscope (14, 16). The potential programs
` were generated in the computer and applied to
` the device reservoirs through high-voltage relays
` and FUG model HCN 12500 power supplies.
`
` ROH2C CH20R
`
` ROH2C ---Zn H2OR
` ROH2C CH20R
`
` Department of Chemistry and Biochemistry, University
` of Texas, Austin, TX 78712.
`
` *To whom correspondence should be addressed.
`
` R R= COH21
`
` ROH2C CH20R
`
` Fig. 1. Structure of the photoconductor
` ZnODEP.
`
` SCIENCE * VOL. 261 * 13 AUGUST 1993 897
`
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