Silicon-microfabricated diffusion-based optical chemical sensor

Silicon-microfabricated diffusion-based optical chemical sensor

ELSEVIER Sensors and Actuators I3 38-39 (1997) 452-457 B CHEMICAL Silicon-microfabricated diffusion-based optical chemical sensor B.H. Weigl *, P...

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ELSEVIER

Sensors and Actuators I3 38-39 (1997)

452-457

B CHEMICAL

Silicon-microfabricated diffusion-based optical chemical sensor B.H. Weigl *, P. Yager Centerfor

Bioengineering,

University of Washing!on, Box 3.72141, Seattle, WA 98195, UXA

Abstract A silicon-microfabricated flow structure is presented that can be used to detect chemical concentrations optically in complex sample solutions. The principle is exemplified by determining the pH of a sample using a fluorescent pH indicator. The flow behavior of liquids in microstructures differs significantly from that in the macroscopic world. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without any mixing other than diffusion. On the other hand, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients, which are a function of particle size. We have designed Tshaped silicon channels, in which a sample solution and a receptor solution containing the indicator dye are joined in the T-connection. The two streams flow next to each other without turbulent mixing until they exit the structure. Small molecules and ions diffuse rapidly acrossthe width of the channel, whereas larger molecules diffuse more slowly. Larger particles such as blood cells show no significant diffusion within the time the two flow layers are in contact with each other. These analyte molecules diffuse into the adjacent acceptor stream with the fluorescent indicator dye. The fluorescence properties of the indicator are a function of the concentration of the analyte molecules in the interaction zone between the two streams and can be monitored. Keywords:

Diffusion;

Microfabrication;

Optical chemical sensors; Silicon

1. Introduction Using tools and technologies developed in the semiconductor industry, it has recently becomepossible to manufacture flow structures and fluid-handling

devices with feature

sizes in the low micrometer range [l-3], Such devices can bemassproduced in silicon by techniquessuchasanisotropic etching or reactive ion etching. It is expected that analytical devices based on silicon-microfabricated devices will soon be in widespread use [ 3,4]. Such devices offer many advantages over traditional analytical devices, such as low sample consumption and cheap unit prices, but also have distinctive properties inherent to their small dimensions. Relevant properties for the sensor presented in this paper are: (a) fiow in

microchannels is laminar; (b) in small channels diffusion is an efficient processfor mixing fluids; and (c) particles can be separatedby diffusion according to their size (diffusion coefficient). 1.1. LaminarJlow Most microfabricated fluid systems operate under low Reynolds number conditions [ 21. The Reynolds number is * Corresponding

author. Phone: + 1206 616 3129.

0925-4005/97/$17,00 0 1997 Elsevier Science S.A. All rights reserved PIISO925-4005(96)02120-X

the ratio of inertial forces to the viscous force. Since in small channels only small liquid massesare moved and flow velocities are typically low, this ratio is small for a given viscosity. Therefore, an aqueousflow streamin amicrochannel behaves like a more viscous oil-like streamin a macroscopicchannel. It is therefore possible to let two aqueousstreamsof the same viscosity flow next to each other for unlimited time without turbulent mixing [ 25-71. 1.2. DifSusion-basedmixing and separation Molecules, ions, and small particles diffuse rapidly over typical microfabricated dimensions. The distanceI that a typical spherical particle will diffuse in time t is I = (Dt) o.5,D is the diffusion coefficient, which is, for a given temperature and solvent viscosity, a function of the diameter of the particle. For example, at room temperature in aqueoussolution, a spherical molecule with a molecular weight of 330 takes 0.2 s to diffuse 10 pm, whereasa bead with a diameter of 0.5 pm takes about 200 s to cover the same distance [S-10]. This effect can be used to separatemolecules and particles according to their size [ 111, and is particularly useful in microfabricated devices [ 2,12-N].

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1.3. Di$fusion-based detection Fluorescent indicator dyes for pH, ions, and biomolecules are in widespread use in research laboratories and in some commercial instruments. They usually show excellent sensitivity, but also some cross-sensitivities to other analytes. Fluorescence measurements are also very sensitive to scattering particles in the sample. Therefore, in order to determine an analyte optically in complex matrices such as blood, the solid components have to be removed by centrifugation or filtering prior to the measurement.In thepresentedflow structures, we rely on both laminar flow and diffusion-based mixing and separation to detect a single analyte in a complex sample. In a T-shaped silicon channel, a sample solution and a receptor solution, containing the indicator dye, are joined in the T-connection. The two streamsflow laminarly next to each other until they exit the structure. Small ions and molecules such as Hf and Na’ diffise rapidly acrossthe diameter of the channel and into the receptor solution, whereas larger molecules and ions such asthe dye anion diffuse more slowly. Large particles such asblood cells show no significant diffusion within the time the two flow layers are in contact with each other. The analyte molecules or ions cause the indicator dye in the receptor stream to changeits fluorescence properties. The change in fluorescence intensity or emission maximum is then detected and used as a measure for the analyte concentration. This device is the subject of a US Patent Application from the University of Washington [ 201. 2. Experimental

2.1. Silicon processing Silicon micromachining was performed in the facility of the Washington Technology Center. Two different types of diffusion-based flow devices, which we will call the T-Sensor and Viewport-T-Sensor, were manufactured. The fabrication techniques have been describedelsewhere [ 21. In the following we describe the process in abbreviated form (see also Fig. 3). First, two photographic masks were made for each flow device, one for the fluid connection holes and one for the channels, Fig. 1 shows the mask of the T-Sensor, and Fig. 2 the mask layout of the Viewport-T-Sensor. Photoresist was spun on 3 inch silicon wafers of 330 p,rn thickness that had about 1 p,rn of wet thermal SiO, on them. The mask for the channels and a silicon wafer were aligned and exposed using a contact aligner. The wafer was developed.Bluetack tape was applied to the backsidesofthe wafers to protect the oxide from the oxide etch. The wafer was then immersed in a buffered oxide etch to remove 600 nm of the unprotected oxide. The Bluetack tape was removed, and the photoresist was rinsed off in acetone. The wafer was again coated with photoresist, the fluid-connection-hole mask was aligned, and the mask was exposed.Again, Bluetack tapewas

453 mixture exit port

indicator inlet port

Fig. 1. The mask layout of a T-Sensor reflecting the widest portions of channels etched into a Si wafer. The inlet port channels are 200 pm wide, and the detection channel is 400 pm wide. The depth of the complete structure is 50 pm.

+

+

mixture exit port

+

Fig. 2. The mask layout of a Viewport-T-Sensor. The buffer solution of pH 5 ( = sample) comes from the left inlet, and a weakly buffered indicator dye solution (pH 9) enters from the right. Portions of the receptor stream which contains the indicator dye are continuously taken out of the channel at various locations into the viewports, which have an area of several mm’.

applied, and all oxide was removed in oxide etch as before. Silicon etching was done in EPW-F etch. This etch attacks the { 100} planes of silicon at a rate of 100 p,m h- ‘. First, the fluid connection holes were etched through the wafer, then the residual 400 nm of oxide were removed by an oxide etch. Finally, the flow channels were etched about 50 p,rn deep. After cleaning, a 100 pm Pyrex glass wafer was anodically bonded to the silicon wafer at 485°C and a potential of 800 V. The Pyrex-silicon wafers were diced into individual devices by scratching the silicon with a diamond tip and gently breaking the wafers along thoselines. Hose connectors consisting of a stainless-steeltube pressedinto apolycarbonate sleeve were glued onto the fluid connection holes with epoxy glue. The processis shown schematically in Fig. 3. 2.2. Fluid controls The hose connectors were connected to a T-piece, which was connected with 0.28 mm ID Teflon tubing to a sampleinjection syringe and a pressuremanifold rack. The pressure

B.H. Weigl, P. Yager/Sensors

454

STEP

1: UNCOATED

SftlCON

STEP 2: TOP.SIDE THROUGH HOLES

ETCHING

STEP FLOW

3 BO,TOM.SIDE CHANNEi

WAFER

prepared as follows: 1 mg of the fluorescent pH indicator Carboxy-SNAFL 1 (Molecular Probes, Eugene, OR) was dissolved in 2 ml DMSO. This dye has a pK, of 7.4, and a useful dynamic range from pH 6.5 to 8.5. It had a yellow fluorescence emission in its acid form, and a red emission in its base form when excited at 4 80 nm. For the Viewport-TSensor, 200 p,l of this solution were mixed with 2 ml of a 0.05 mM HEPES buffer solution of pH 9. For all other experiments, the acceptor stream was a mixture of 200 p,l of the DMSO-Carboxy-SNAFL 1 solution with 2 ml of a 0.05 n&I HEPES buffer solution of pH 5.5. For the proof-of-principle experiment and the Viewport-TZiensor experiment, a 10 mM HEPES buffer solution of pH 9 was used as the sample. For the pH calibration experiment, the samples were 10 mM HEPES buffer solutions adjusted to the following pH values: 7.2,7.4, 7.6,7.8, and 8.0.

OF

ETCHiNG

STEP 4. Al-fACXMENTOF ONTOP OF FLOW CHANNEl (ANODE BONDINGI

and Actuators B 38-39 (1997) 452-457

GLASS

STEP 5 ATTACHMENT OF HOSE CONNECTORS TO THE TOP SIDE THROUGH HOLES f-1

OF

WAFER bL

OF

hid

17

Fig. 3. Schematic of the silicon microfabrication process used for manufacturing the T-Sensor and the Viewport-T-Sensor.

differences between inlets and outlets of the channels were generated with water-filled glass tubes that were positioned so that the pressure drop was about 50 mm of water (5 mbar). For some experiments, the pressure difference was varied between 10 and 150 mm to demonstrate the effect of flow rate on the sensing mechanism. Since the volumes of the glass tubes were very large compared to the total sample volume, the pressure differential was practically constant during the experiment.

3. Results and discussion Fig. 4 describes the expected behavior of a T-Sensor. The sample is brought to the junction through one port (right) and a solution containing an indicator dye through the second port (left). The two streams are joined in the T and flow parallel to each other towards an exit port (top), Due to the low Reynolds number in such small channels, no turbulenceinduced mixing occurs and the two fluids flow parallel to each other without mixing. However, because of the short distances involved, diffusion does occur, visible only perpen-

2.3. Optical monitoring A Zeiss ICM 405 inverted microscope was equipped with an Omega Optical XF34 fluorescent filter set (h,, = 480 nm, h,, > 510 nm) and a Zeiss 35 mm camera with 400 ASA Kodak color negative film. The silicon device was attached to the stage of the microscope with adhesive tape, and connected to the fluid controls. Photographs were taken of the flow channel for each sample with an exposure time of 1 s. The film was developed using no color or exposure correction, and the same filtering was used for each image. The photographs were scanned using a Silverscan III color scanner, and, for the purpose of publishing, converted into grayscale images using the software package Adobe Photoshop 3.0. I’

2.4. Sample preparation

Indicator 01 Indicator beads in weakly buffered

All chemicals used were obtained from Aldrich Chemical Company, unless otherwise noted. The acceptor solution was

solution Fig. 4. Schematic of flow and diffusion within the T-Sensor,

sample soiutlon, contammg particles and soluble components

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dicular to the flow direction. At the particular pH values used in these experiments, mainly OH- ions diffuse to the left into the indicator stream and, eventually, become uniformly distributed across the width of the channel. The indicator also diffuses in the opposite direction, but more slowly, because of its greater diffusion coefficient. If this change in local concentration of the indicator is a problem in some applications, its diffusion rate can be made arbitrarily small by immobilization on polymers or beads. Small ions such as H+ and Na+ diffuse rapidly across the diameter of the channel, whereas larger molecules such as the dye, sugars, proteins, etc., diffuse more slowIy. Larger particles such as blood cells show no significant diffusion within the time the two flow layers are in contact with each other, The smaller sample components equilibrate close to the T-joint, whereas larger components equilibrate further up in the channel. Furthermore, as the indicator has a particular half-saturation concentration (pK, in the case of a pH dye), a front of indicator dye color or fluorescence change exists as diffusion proceeds up the channel, as shown by the curved line within the channel above. The location and curvature of the front is a sensitive indicator of the concentration of the analyte; its ‘resting location’ can be adjusted by changing the flow speed and channel width. Although this is a flow system, the physical location of the front in the channel for a given analyte stays the same over time as long as the flows are constant and the sample unchanged. The analyte concentration can be determined by monitoring the indicator signal at various locations along the channel, after substantial equilibration, for example with a multi-element detector. An experiment was conducted for the device shown in Fig. 4. A buffer solution of pH 9 entered from the right port. A weakly buffered indicator dye solution of pH 5.5 entered from the left port, The buffered solution (pH 9) appears as a dark (clear) fluid. Fig. 5 shows grayscale versions of color fluorescence microscope photographs of a T-sensor featuring a buffer solution of pH 9 (right inlet) in a dynamic flow equilibrium (at two different pressure differentials) with a receptor solution containing an indicator dye in a weakly buffered solution of pH 5 (left inlet). The pressure difference between inlet and outlet ports was 30 mm water for the experiment shown in Fig. 5 (top), and 70 mm for the experiment in Fig. 5 (bottom), thereby showing the influence of flow speed on the behavior of the sensor. The light-gray section on the left (originally yellow) of the channels represents the acid form of the dye. The darker-gray wedge in the middle (originally orange-red) shows the interaction zone between analyte and indicator molecules. The black (colorless) stripe on the right is the sample stream. Some of the advantages of the T-Sensor concept are: (a) analytes can be determined optically in turbid and strongly colored solutions, such as blood, without the need for prior filtering or centrifugation; (b) cross-sensitivities of indicator dyes to larger sample components (a common problem) can be avoided;

455

Fig. 5. Fluorescence micrographs of a T-sensor in which a buffer solution of pH 9 (right inlet) is flowing into the device, and a weakly buffered indicator dye solution (pH 5) enters from the left. Note the distinct conversion of the dye from one form to the other as diffusion proceeds. (Top) The T-sensordtiven by a pressure differential of 30 mm water column. (Bottom) The same experiment with a pressure differential of 70 mm water column.

(c) the indicator can be kept in a solution in which it displays its optimal characteristics (e.g., cross-sensitivities to pH or ionic strength can be suppressed by using strongly buffered solutions) ; (d) measuring the fluorescence of the indicator dye at several locations along the channel can compensate for some remaining cross-sensitivities; (e) the charme can be made relatively wide, which makes it easier to measure the indicator fluorescence with simple optics, and less likely for particles to clog the channel; (f) the steady-state nature of this method makes longer signal integration times possible; (g) the location of the diffusion boundary gives information about flow speed and/or sample concentration. Another design based on the same principle, the ViewportT-sensor, has been fabricated and tested (see also the mask layout in Fig. 2). Please note that in the experiment described in Fig. 6 the indicator stream comes from the right T-leg, and is a solution of indicator dye in a low-ionic-strength buffer of pH 9. The sample stream, which is introduced from the left, is a 100 mM buffer solution of pH 5.5. Several portions of the receptor stream that contains the indicator dye are continuously taken out of the channel at various locations. These portions flow into relatively wide channels in the silicon wafer that serve as viewports. The viewport closest to the T-joint contains mainly undisturbed dye solution, whereas the viewport closest to the outlet contains the acceptor stream completely equilibrated with the sample. The viewports in between contain the acceptor stream in various degrees of

B.H. Weigl, P. Kager/Sensors

and Actuators B 38-39 (1997) 452457

Fig. 6. Fluorescence micrograph (composite image) of the experiment described above. The dark gray color in the lowest viewport represents the red color of the baseform of the undisturbed indicator dye, whereas the light gray (yellow-green color) of the higher viewports represent the acid form of the dye.

equilibration with the sample components.The closer it is to the T-joint, the more likely the viewport is to contain only small ions from the sample. Fig. 6 shows a fluorescence micrograph (composite image) of the experiment described above. The dark-gray shade in the lowest viewport representsthe red color of the base form of the undisturbed indicator dye. The light gray (yellow-green color) of the higher viewports representsthe acid form of the dye, after the pH of the receptor streamwas altered from basic to acid after diffusion-based equilibration. In addition to the advantages of the T-Sensor described above, the Viewport-T-Sensor lends itself to simple referencing techniques. Due to their size (several mm2), the integral fluorescenceintensity of each viewport at one or more wave-

lengths caneasily be monitored through afluorescencemicroscope, or directly with a photodetector. In the easiest case (with an indicator dye showing no cross-sensitivity to other sample components) the intensity ratio between selected viewports gives a measurementvalue largely independentof dye concentration and excitation. light intensity. Measuring at more than one viewport increases the redundancy and therefore the measurementaccuracy. In the case of cross-sensitivity of the indicator to larger sample components (e.g., larger biomolecules such as albumin) , this interference can be referenced out by comparing the ratios of the different viewports. The viewports closer to the T-joint will contain mainly smaller sample components, whereasthe viewports further up ihe channel will also contain larger particles. In order to demonstratethe ability of the T-Sensor tomeasure the concentration of an analyt.e,buffer solutions with five different pH values between7.2 and 8 were used assamples. The experiment was performed in the sameway asthe proofof-concept experiment describedabove,Fig. 7 shows a grayscale version of the five photographs taken when the sample was in the channel. The photographs show a section of the T-channel. The top part (white-hght gray, originally yellow) shows the acid form of the dye. The middle section (from left to right, pH 8.0 to pH 7.2, Clarkgray over gray to light gray, originally deep red over orange to yellow) shows the interaction zone between the analyte (OH- and H+) and the indicator dye. The bottom part (black) shows the colorless samples.

4. Conclusions

The devices presented here allow the optical monitoring of an analyte concentration in a very small volume of a complex sample. By coupling such T-Sensors or Viewport-TSensorsto CCDs, or to multi-wavelength detectors,we expect that it will be possible to determine accurately the concentration of a multitude of analytes in bodily fluids such asblood. Since such devices can be easily mass manufactured, they have potential for use in the med.icaldiagnostic field.

Fig. 7. Grayscale version of five photographs taken when samples with pH values of 8.0,7.7,7.6, 7.4, and 7.2 were in the channel. The photographs show a section of the T-channel. The top part (white-light gray, originally yellow) shows the acid form of the dye. The middle section (from left to right (pH 8.0 to pH 7.2), dark gray over gray to light gray, originally deep red over orange to yellow) shows the interaction zone between the analyte (OH- and H+ ) and the indicator dye. The bottom part (black) shows the colorless samples.

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and Actuators B 38-39 (1997) 452-457

Acknowledgements We thank Dr. M. Ho11 for designing the fluid-control unit and for modelling the flow patterns in the T-Sensor, which will be reported elsewhere, and D. Schutte for help with microfabrication of the flow structures. This work was supported by Senmed Medical Ventures, ARPA (grant # DAMD17-94-J-4460)) and the Washington Technology Center.

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Biographies Bernhard H. Weigl received his MSc. in chemistry in 1990, and his Ph.D. on optochemical sensor modules for CO2 in 1993, both from Karl-Franzens-University Graz, Austria. After a postdoctoral stay at the Optoelectronic Research Centre, University of Southampton, UK, he is now senior research fellow at the Center for Bioengineering, University of Washington, Seattle, USA, designing microfabricated optical modules for the determination of blood parameters. Paul Yager is professor in the Center for Bioengineering at the University of Washington in Seattle. He holds adjunct appointments in chemistry and chemical engineering. He received his Ph.D. in chemistry from the University of Oregon in 1980, and then went to the Naval Research Laboratory in Washington, DC, first for a NRC Postdoctoral Pellowship, and then as aresearch chemist. He joined the faculty at the University of Washington in 1987.