A Microfluidic Device with an Integrated Waveguide Beam Splitter for

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Anal. Chem. 2003, 75, 4931-4936

A Microfluidic Device with an Integrated Waveguide Beam Splitter for Velocity Measurements of Flowing Particles by Fourier Transformation Klaus B. Mogensen,† Yien C. Kwok,‡,§ Jan C. T. Eijkel,‡,| Nickolaj J. Petersen,†,⊥ Andreas Manz,‡ and Jo 1 rg P. Kutter*,†

Mikroelektronik Centret (MIC), Technical University of Denmark (DTU), DK-2800 Lyngby, Denmark, and Department of Chemistry, Imperial College, London SW7 2AY, U.K.

A microfabricated capillary electrophoresis device for velocity measurements of flowing particles is presented. It consists of a 1 × 128 planar waveguide beam splitter monolithically integrated with an electrically insulated fluidic channel network for fluorescence excitation at multiple points. Stray light rejection structures are included in order to suppress unwanted light between the detection regions. The emission pattern of particles passing the detection region was collected by a photomultiplier tube that was placed in close proximity to the channel, thereby avoiding the use of transfer optics. The integrated planar waveguide beam splitter was, furthermore, permanently connected to the light source by a glued-on optical fiber, to achieve a robust and alignment-free operation of the system. The velocity was measured using a Fourier transformation with a Shah function, since the response of the light array was designed to approximate a square profile. Deviations from this response were observed as a result of the multimode nature of the integrated waveguides. During the past decade, the ability to separate and characterize particulate species of different origins has become increasingly more important. A wide range of techniques, such as free-flow electrophoresis,1 capillary electrophoresis,2,3 dielectrophoresis,4 and various types of chromatography5-7 has been used for this purpose. Mobility measurements are a versatile tool, because * Corresponding author. E-mail: [email protected]. † Technical University of Denmark. ‡ Imperial College. § Natural Sciences Academic Group, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616. | EL/BIOS, Twente University, Postbus 217, 7500 AE Enschede, The Netherlands. ⊥ Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6142. (1) Slivinsky, G. G.; Hymer, W. C.; Bauer, J.; Morrison, D. R. Electrophoresis 1997, 18, 1109-1119. (2) Radko, S. P.; Chrambach, A. Electrophoresis 2002, 23, 1957-1972. (3) Mehrishi, J. N.; Bauer, J. Electrophoresis 2002, 23, 1984-1994. (4) Gascoyne, P. R. C.; Vykoukal, J. Electrophoresis 2002, 23, 1973-1983. (5) Kok, W. T.; Stol, R.; Tijssen, R. Anal. Chem 2000, 72, 468A-476A. (6) Tijssen, R.; Bos, J.; van Kreveld, M. E. Anal. Chem. 1986, 58, 3036-3044. (7) Chmela, E.; Tijssen R.; Blom, M. T.; Gardeniers, H. J. G. E.; van den Berg, A. Anal. Chem. 2002, 74, 3470-3475. 10.1021/ac034427a CCC: $25.00 Published on Web 08/16/2003

© 2003 American Chemical Society

information as diverse as the size distribution of polymers,6 the quantity of surface charge of cells,3 and adsorption of polymeric surfactants onto microparticles8 can be obtained. Noninvasive optical techniques prevail for determination of the migration rate, since disruption of the system is avoided. Laser doppler velocimetry,9,10 particle image velocimetry,11,12 and fluorescence correlation spectroscopy13 are among the commonly used techniques. Recently, various multiple-point detection schemes have been presented for particle velocity measurements in capillary electrophoresis.13-15 Detection at multiple points is advantageous compared to traditional single-point detection, because measurements on a continuous stream of particles can be performed, thereby avoiding the necessity to define and inject a sample plug. The vast majority of techniques presented so far relied on extensive use of free-space optical elements, such as lenses, mirrors, and beam expanders13-15 that are carefully aligned in an optical setup. Such systems are difficult to miniaturize further and very sensitive to shock and vibrations. The motivation of this study was to overcome the packaging and stability problems related to the use of free-space optical elements for detection by monolithic integration of a planar waveguide beam splitter for multiple-point fluorescence excitation. This allows alignment-free operation, because the chip can be directly connected to the light source by glued-on optical fibers. Such a system can therefore potentially be operated by a nontechnical user and should also have significantly reduced packaging costs. A microfluidic device with an integrated 1 × 16 planar waveguide beam splitter has recently been presented,16 but parallel detection at multiple points was not demonstrated. Other studies (8) Huff, B. V.; McIntre, G. L. J. Microcolumn Sep. 1994, 6, 591-594. (9) Fourest, B.; Hakem, N.; Perrone, R.; Guillaumont, R. J. Radioanal. Nucl. Chem. 1996, 208, 309-318. (10) Dearie, H. S.; Spikmans, V.; Smith, N. W.; Moffatt, F.; Wren, S. A. C., Evans, K. P. J. Chromatogr., A 2001, 929, 123-131. (11) Meinhart, C. D.; Wereley, S. T.; Santiago, J. G. Exp. Fluids 1999, 27, 414419. (12) Klank, H.; Goranovic, G.; Kutter, J. P.; Gjelstrup, H.; Michelsen, J.; Westergaard, C. H. J. Micromech. Microeng. 2002, 12, 862-869. (13) Sonehara, T.; Kojima, K.; Irie, T. Anal. Chem. 2002, 74, 5121-5131. (14) Kwok, Y. C.; Jeffery, N. T.; Manz, A. Anal. Chem. 2001, 73, 1748-1753. (15) McReynolds, J. A.; Edirisinghe, P.; Shippy, S. A. Anal. Chem. 2002, 74, 5063-5070.

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on buried channel planar waveguides integrated with a microfluidic channel network have previously only been used for oneand two-point measurements.17-21 One of the multiple-point detection schemes used in this study was previously introduced by Crabtree et al.22 It was denoted Shah convolution Fourier transform detection (SCOFT) and utilizes a Shah convolution of an emission signal generated during operation of the chip. The time-domain signal is subsequently deconvoluted by performing a Fourier transformation (FT). A Shah function consists of equally spaced δ-functions. Such an emission profile was generated on-chip by illuminating a channel segment through a slit array. Excitation of the analyte fluorescence was hence obtained at regularly spaced regions along the microfluidic channel. This resulted in a periodic emission pattern when fluorescent particles or bands of fluorescent solutes moved along the channel. The frequency components, which are proportional to the bead or band velocities, were extracted by performing a Fourier transformation of the signal. In another approach, the convolution function was defined by the collection optics rather than the excitation optics by clever use of a charge-coupled device.15 The strength of this approach is that the individual pixels can be controlled to generate almost any desired convolution function, which greatly increases the versatility of the system. A major disadvantage is that the sampling frequency is low (28 Hz in ref 15) compared to the megahertz range typically achieved with photomultiplier tubes,23 limiting its use for higher frequencies/bead velocities. An alternative data analysis scheme is based on wavelet transformation of the time-domain signal.24 This approach is advantageous compared to Fourier transformation, because the frequency components can be extracted as a function of time, thereby providing an additional dimension of information. EXPERIMENTAL SECTION Design and Fabrication of the Microfluidic Chip. The design of the device is shown in Figure 1. Each chip was about 4.5 cm × 5.0 cm, which means that two devices could be present on a 4-in. wafer. The microfluidic channel network consisted of an injection cross and a separation channel with a total of four fluidic reservoirs. All channels were 50 µm wide and 13.7 µm deep. Two 1 × 128 planar waveguide beam splitters were present along the separation channel for multiple-point fluorescence excitation. Figure 2A shows a closer view of a region on the mask design where the waveguides meet the separation channel. The (16) Ruano, J. M.; Glidle, A.; Cleary, A.; Walmsley, A.; Aithison, J. S.; Cooper, J. M. Biosens. Bioelectron. 2003, 18, 175-184. (17) Ruano, J. M.; Benoit, V.; Aitchison, J. S.; Cooper, J. M. Anal. Chem. 2000, 72, 1093-1097. (18) Pandraud, G.; Koster, T. M.; Gui C.; Dijkstra, M.; van den Berg, A.; Lambeck, P. V. Sens. Actuators, A 2000, 85, 158-162. (19) Mogensen, K. B.; Petersen, N. J.; Hubner, J.; Kutter, J. P. Electrophoresis 2001, 22, 3930-3938. (20) El-Ali, J.; Mogensen, K. B.; Nielsen, I. R. P.; Kutter, J. P.; Telleman, P.; Wolff, A. In Proceedings of Micro Total Analysis Systems 2002; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2002; pp 260-262. (21) Lee, G. B.; Lin, C. H.; Chang, G. L. Sens. Actuators, A 2003, 103, 165170. (22) Crabtree, H. J.; Kopp, M. U.; Manz, A. Anal. Chem. 1999, 71, 2130-2138. (23) Photomultiplier Tubes and related products, June 2002; Hamamatsu Photonics K. K., 2002. (24) Eijkel, J. C. T.; Kwok, Y. C.; Manz, A. Lab Chip 2001, 1, 122-126.

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Figure 1. Design of the SCOFT device consisting of an integrated planar waveguide beam splitter, stray light rejection structures, and an electrically insulated microfluidic channel network.

Figure 2. (A) Detailed view of the chip design where the waveguides intersect the microfluidic channel. (B) Symmetric planar waveguide Y-junction with stray light rejection structure.

waveguides had a width of 24 µm and were spaced along the channel with a center-to-center distance of 100 µm. The 128 waveguides in a single beam splitter spanned a total length of 12.8 mm, and the two beam splitters in the design covered a combined region of 25.6 mm. The trenches along either side of the waveguides were 15 µm wide. The microfluidic channel was positioned 25 µm from the trenches that defined the waveguides. The distance from injection to the first waveguide was 3.2 mm, while the distance from the last waveguide to the waste reservoir was 33 mm. The lengths of the three inlet channels (to the injection cross) were 2.0, 3.0, and 4.0 cm, respectively. The gray area (Figure 2A) is the region where the lid is bonded to the wafer. It contains waveguides and bonding regions to avoid leakage of liquid into the trenches along the waveguides.25 The chip fabrication required two mask layers that were designed using the program Prometheus 4.0 (C2V, Enschede, The Netherlands). The first mask consisted of planar waveguides and bonding regions, while the second mask consisted of fluidic channels and stray light rejection structures. The multimode waveguide beam splitters were made by serial connection of symmetric Y-junctions. A single Y-junction is shown in Figure 2B. The waveguide bends were made from two segments of a circle with a constant radius of 18 mm. The splitting was performed 7 (25) Friis, P.; Hoppe, K.; Leistiko, O.; Mogensen, K. B.; Hu ¨ bner, J.; Kutter, J. P. Appl. Opt. 2001, 40, 6246-6251.

times, which resulted in 27 ) 128 excitation points. The overall length of 44 mm of the beam splitters was determined by the channel segment that was to be illuminated (12.8 mm) and by the waveguide bend radius. As a rule of thumb, a value for the ratio between the bend radius and the half-waveguide width of more than 1000 for a refractive index step of ∼0.02 was used to ensure negligible radiation loss in the bend.26 In this design, the ratio was chosen to 18 000 µm/12 µm ) 1500 in order to be on the safe side. Stray light rejection structures were positioned between the two waveguide branches of each Y-junction. They consisted of channels etched into the waveguide glass layers to suppress transmission of stray light (see also Results and Discussion). The device was fabricated by using surface micromachining processes, such as plasma-enhanced chemical vapor deposition and reactive ion etching of glass. The fabrication process is described in more detail in ref 19. The buried channel waveguides consisted of three layers of glass, denoted waveguide buffer (n ) 1.458), core (n ) 1.480), and cladding (n ) 1.458). The refractive index of the core layer was increased by co-doping with nitrogen. Wave guidance was therefore obtained in the core layer by means of total internal reflectance. The depth of the microfluidic channel was 13.7 µm. The waveguides had a thickness of 7.5 µm and were located 1.1 µm above the bottom of the fluidic channels, to ensure that all the excitation light guided in the core layer would be coupled into the channel. A 16.9-µm-thick layer of thermally grown silicon dioxide was present between the silicon substrate and the microfluidic channels for electrical insulation.19 The stray light rejection structures were etched to a depth of 23.4 µm. This was done during the etching of the waveguide pattern and again during etching of the microchannels. There was a layer of 7.2 µm of SiO2 left underneath the blocking structures toward the silicon substrate. The stray light rejection structures were never exposed to any electrical fields. Reagents and Solutions. A Tris-borate-EDTA (TBE) buffer solution was purchased from Sigma-Aldrich (Steinheim, Germany) and diluted to a 1× concentration with deionized water (18 MΩ‚ cm, Millipore SA, Molsheim, France) to give a pH of 8.8. A 10 mM tetraborate buffer solution of pH 9.1 was prepared from disodium tetraborate decahydrate (Merck, Darmstadt, Germany). Amine-modified, yellow-green fluorescing polystyrene microspheres with diameters of 1.0 (505/515, F-8765) and 4.0 µm (505/515, F-8859) were purchased from Molecular Probes Europe BV. Two solutions of the 1.0-µm microspheres were prepared by 1000- and 25 000-fold dilution in a 1× TBE buffer solution, respectively. The 1000-fold dilution of the 1.0-µm-diameter microspheres corresponded to about 10-30 beads in the detection region at any given time, while the 1000-fold dilution of the 4.0-µm-diameter beads in 10 mM borate buffer solution corresponded to single beads in the detection region. Fluorescein was purchased from Molecular Probes Europe BV and prepared to a concentration of 170 µM using the 10 mM borate buffer solution. Apparatus. The 488-nm line from an argon ion laser (150 mW, model T543R-AP-A01, Melles Griot) was coupled into the waveguide beam splitter by an optical fiber (50-µm core diameter, (26) Snyder, A. W.; Love, J. D. Optical Waveguide Theory, 1st ed.; Chapman & Hall, New York, 1991.

FVP050055065; Polymicro Technologies) that was permanently attached to the waveguide end face by glueing (NOA68, Thorlabs). A 5.1-cm head-on photomultiplier tube (R550 PMT, E1198-11 socket, Hamamatsu Photonics, Middlesex, U.K.) equipped with a fluorescein emission band-pass filter (520DF15, Omega Optical) and a high-pass filter (LWP-25-515 Delta Light & Optics) was used for optical detection of the fluorescence signal. The detector was placed directly on top of the channel, which enabled a 90° separation between the excitation and collection axes. No transfer optics were used for collection of the fluorescence signal, since the PMT could be positioned in close proximity to the channel. This also helped in avoiding alignment of the detector. Aluminum foil was wrapped around the filters and the chip to reduce collection of unfiltered light. Data acquisition was carried out at 1000 Hz with a 6-dB low-pass filter set to 300 Hz. Experimental Procedure. The daily chip preparation consisted of flushing the channels with a 1.0 mM NaOH solution for 2 min followed by a 5-min rinse with the buffer solution used in the subsequent experiment. All the reservoirs were filled with the sample solution, and Pt electrodes were placed in the outlet and in one of the inlet reservoirs. A home-built power supply that was capable of delivering an output voltage of 4 kV was used. Data collection and control of the power supply was done by software written in LabView 6i (National Instruments). Data were collected for 4 min. Data Analysis. The Fourier and wavelet transformations of the time-domain signals were done in MatLab 6.0 (The MathWorks Inc., Natick, MA). No apodization function was used for the Fourier transformations. Visualization of the frequency components of the data set was obtained by calculation of the magnitude spectrum using the formula

FTv,magnitude ) ((FTv,real)2 + (FTv,imaginary)2)1/2

(1)

FTv,real is the real part of the Fourier transformed time-domain signal, while FTv,imaginary is the imaginary part. Wavelet transformations of the data sets were performed in order to obtain time information in addition to frequency information. The following function for the wavelet was used:

y ) exp(-x32/2) cos(200x)

(2)

This is a type of function similar to that used by Eijkel et al.24 The signal-to-noise ratio of the peaks in the time domain was calculated by dividing the difference between the peak height and the baseline level with the standard deviation of the baseline signal measured over a period of 1 s (without beads). The S/N of the peaks in the frequency domain of the Fourier transforms was calculated by dividing the difference between the peak height and the baseline level by the standard deviation of a 2-Hz-wide baseline signal (without beads) at the same frequency as the peak. RESULTS AND DISCUSSION Waveguide Properties. The performance of a planar waveguide beam splitter without integrated stray light rejection structures was initially tested. The optical output is shown in Figure 3. This device was fabricated only to investigate the performance of the Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

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Figure 3. Optical output from a planar waveguide beam splitter without integrated stray light rejection structures. The stray light in the middle of the device originates from coupling loss between the optical fiber and the waveguide beam splitter. It should be noted that the waveguides on this particular device were cleaved accidentally at a position where they had a small deviation from the 90° angle toward the interface, which is the reason for the apparent change in the spacing between the light rays.

Figure 4. Detailed view of the detection channel being illuminated through an integrated 1 × 128 waveguide beam splitter (image taken from the center section of the waveguide array). The light paths were visualized by filling the channels with a 170 µM fluorescein solution. Excitation was achieved by 488-nm light from an argon ion laser that was coupled through a fiber into the waveguide beam splitter. No stray light between the illumination points could be observed by visual inspection.

beam splitter, so no microfluidic channel network was integrated. The end of the waveguide beam splitter was immersed in a fluorescein solution to visualize the light paths. A significant amount of stray light is present between the splitted waveguide branches, which clearly shows the necessity to integrate light blocking structures. The stray light mainly originates from coupling loss between the input fiber and the planar waveguide beam splitter. The blocking structures were etched in the same processes as used for fabrication of the waveguide and microfluidic channel patterns and could therefore be included in the final devices without any additional processing (Figure 1). The properties of the integrated waveguides in the finished devices were investigated by visual inspection through a microscope. The result is shown in Figure 4, where the channel network was filled with a 170 µM fluorescein solution. Illumination in well-defined regions with a spacing of 100 µm (corresponding to the distance between the waveguides), can be seen in the microscope pictures. Stray light between the illumination points was not observed. Inspection of the integrated stray light rejection structures showed that transmission of unguided light between the waveguides was significantly reduced. Figure 5 shows a close view of two previously split waveguide branches. The lines between the waveguides are the stray light rejection structures, which consist of trenches etched into the glass layers. 4934 Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

Figure 5. Microscope picture of stray light rejection structures between two waveguides. Unguided light is scattered out of the plane of the structure and hence will not reach the detection channel.

Figure 6. Trace from a single 4.0-µm-diameter bead passing the last 18 detection points of the beam splitter (E ) 120 V/cm). The difference in the intensities is due to scattering centers in the waveguide glass layers. The inset figure is an expanded view from 18 to 24 s to show the fine structure of two peaks.

The bright spot in the middle of the blocking structure to the right indicates that light is coupled out of the plane of the device. An indirect measurement of the excitation profile of the multiple excitation points was furthermore carried out by electrokinetically moving a single 4.0-µm-diameter fluorescing bead past the detection region. Distinct peaks corresponding to the bead passing the detection zones are clearly seen in Figure 6. The peaks shown represent only that part of the signal trace originating from the last couple of illumination points of the waveguide array. This allows individual peaks to be distinguished for evaluation of the performance of the individual waveguides and the integrated array. It can be seen that a stable baseline is achieved, between the signal peaks as well as after the bead has left the detection region. This is a direct consequence of the ruggedness of the system. However, a large variation in the excitation power carried by the waveguide branches resulted in different signal-to-noise ratios of the peaks. In the case shown in Figure 6, it varied between about 50 and 500. Single 1.0-µm-diameter microspheres were measured in a similar manner, resulting in signal-to-noise ratios of each peak between about 3 and 30. The variation in peak height is believed to be caused by defects in the waveguides that are introduced due to a nonoptimum fabrication procedure. The most critical fabrication step is the plasma-enhanced chemical vapor deposition of the waveguide core and the cladding layers, since defects are embedded in the glass

during deposition, which can cause excess scattering loss whenever present in a waveguide channel. Defects in the glass layers can significantly be reduced by proper cleaning and conditioning of the plasma chamber before deposition, which is, however, rather difficult to achieve in a research-type, multiuser cleanroom environment. The waveguides were designed to resemble a Shah function (as approximated by a square profile), but rather distorted peaks can be observed in an expanded view of the profiles of two peaks (inset of Figure 6). At first glance, one might suggest that the peak distortion originates from the passage of a cluster of two or more beads rather than from a single bead. This was probably not the case, since great care was taken to ensure that the beads were not clustered and since it was found that the peaks were always distorted. The peak distortion is believed to originate from the multimode nature of the waveguides. In a ray-tracing description, the different modes propagate at different angles, which means that they also will be coupled out of the waveguide under different angles. This is also evident from closer inspection of the light paths shown in Figure 3. A nonuniform intensity profile has previously been observed by Friis27 during quantification of beads with different fluorescence intensities using various types of integrated multimode and single-mode waveguides. In that study, it was found that the effect was much more pronounced the further away the beads were from the end face of the waveguide, because the mode profile of the transmitted light was then better resolved spatially. The problem was avoided by making the waveguides single mode.27 A similar approach was not used in the present study, because the cost for obtaining a better peak shape by only using the fundamental mode to carry the optical power is a significantly reduced signal-to-noise ratio of the detection, since less light can be guided in single-mode waveguides. Instead, a suggested remedy is to place the channel in closer proximity to the waveguides, reducing the spatial resolution of the measurement of the mode profile, which would result in a smoother peak shape. Particle Velocity Measurements. The device was tested for velocity measurements of particles employing Fourier transform detection similar to what has been presented elsewhere14,15 and wavelet transform detection as in the work of Eijkel et al.24 An example of a time-domain signal obtained for 1.0-µmdiameter beads being moved electrokinetically through the detection channel is shown in Figure 7. The electric field strength in this case was 111 V/cm. The number of beads is too high to be able to distinguish the individual peaks from each other; therefore, no directly useful information can be obtained from this representation. However, the amplitudes of the frequency components of the time-domain signal can be extracted by performing a Fourier transformation. The result is shown in Figure 8. The fundamental peak located at 4.1 ( 0.5 Hz corresponds to a bead velocity of 410 ( 50 µm/s. The signal-to-noise ratio of the fundamental peak in this case was 23, which is comparable with the signal-to-noise ratio achieved using the setup presented by Kwok et al.14 The width of the peaks is partly a consequence of a (27) Friis, P., Integrated Optical Detection for Biochemical Microsystems. Ph.D. Thesis, MIC, Technical University of Denmark (DTU), 2002.

Figure 7. A 240-s time-domain signal obtained for 1.0-µm-diameter beads passing the detection region. Conditions: 1× TBE buffer, pH 8.8; electric field strength 111 V/cm.

Figure 8. Magnitude spectrum of a Fourier transformation of the signal from Figure 8. The signal-to-noise ratio of the fundamental peak was 23.

velocity distribution of the beads due to variations in their surface charges and partly governed by the emission profile that was Fourier transformed. A more in-depth investigation on the contribution of the emission profile to the band broadening would require a comparison of the performance of waveguide arrays with different geometries, namely, different waveguide widths and center-to-center spacings. Such systems were, however, not available. The peaks located at multiples of 4.1 Hz are believed mainly to be harmonics of the fundamental peak. A small contribution may come from several beads moving at the same velocity, with a distance corresponding to half of the spacing between the detection regions, because this will result in the same signal as a single bead moving with 2 times the velocity. The microsystem and the described detection technique were further tested by measuring the velocities of beads when the electric field strength in the channel was varied. The results are shown in Figure 9. A linear relation between the bead velocities and the electric field strength is obtained, as expected. The error bars of the measurements are given by the width of the fundamental peak. They are seen to increase for higher field strengths, which can be explained by increased electrophoretic separation of the beads due to variations in their surface charges. The most important issues to address in order to improve the performance of the devices for Fourier transform detection are a Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

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Figure 9. Frequency of the fundamental peak and corresponding bead velocities as a function of the electric field strength.

reduction of the baseline noise and a reduction of the amplitude of the harmonics. One way to do this is by changing the convolution function. McReynolds et al.15 recently showed that going from a Shah function (abrupt changes) to a sine function will make the harmonics disappear and reduce the noise by more than a factor of 2. This can be explained by Fourier theory, since a smooth convolution function (such as a sine function) reduces the amplitude of the side lobes of the FT and also removes the harmonics. It may be possible to obtain a smoother excitation profile along the fluidic channel by inclusion of spreading lenses at the waveguide end faces, thereby emulating a sinusoidal function. An alternative approach is to reject the overtones by numerical means. We expect that this can be done by measuring an instrument function in the spatial domain and transforming to the equivalent spectral representation. The spectral instrument function can subsequently be used to extract the original velocity distribution from the measured signal.28 The time-domain signals can also be analyzed by wavelet transformations in order to obtain the distribution of frequencies as a function of time,24 which could be used for on-line monitoring of a change in the particle’s velocity due to, e.g., binding events. A wavelet transformation is performed by fitting a periodic (28) Karl, J. H. An Introduction to Digital Signal Processing; Academic Press: San Diego, 1989.

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function, denoted a wavelet, to the emission signal generated by the passing particles in order to extract the frequency components of the signal as a function of time. This is explained in more detail in ref 24. A wavelet transformation of the signal from Figure 7 using the wavelet of eq 2 resulted in a band around 4.1 Hz, which was also expected from the Fourier transform shown in Figure 8. This detection scheme can be used in future experiments in cases where the bead velocities are expected to vary while in transit through the system. In conclusion, integrated planar optical waveguides can be used for multiple-point excitation along a microfluidic channel, thereby avoiding the use of free-space optical elements for control of the light paths. This was accomplished with an integrated 1 × 128 planar waveguide beam splitter with stray light blocking structures to avoid illumination between the excitation points. The device was furthermore connected to a light source by the use of a permanently attached optical fiber. This resulted in a robust and alignment-free system. The system was tested by measuring the velocity of 1.0-µm-diameter fluorescing particles at electric field strengths between 111 and 435 V/cm. The measurement scheme in these cases was Shah convolution Fourier transform detection. Wavelet transformations of the time-domain signals were performed in order to track the particle frequencies as a function of time. This detection scheme would be beneficial for future experiments in cases where the bead velocities are expected to vary while the beads are migrating through the system. ACKNOWLEDGMENT Henning Klank from MIC at the Technical University of Denmark is gratefully acknowledged for help with data collection and analysis in LabView and Matlab. This work was funded by the Danish Government’s Scientific Research Council, STVF (Contract 9900683 and frame program Grant 26-00-0220), and EPSRC UK, the U.K. Department of Trade and Industry (DTI), and the Lab-on-a-Chip consortium (LOC).

Received for review April 25, 2003. Accepted July 11, 2003. AC034427A