evanescent molecular tagging technique for electrokinetic...

13th Int Symp on Applications of Laser Techniques to Fluid Mechanics Lisbon, Portugal, 26-29 June, 2006

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Evanescent Molecular Tagging Technique for Electrokinetic Effects

on Velocity Field in the Vicinity of Electrolyte-Glass Interface

Hiroki Fukumura1, Mitsuhisa Ichiyanagi2, Yohei Sato3

1: Department of System Design Engineering, Yokohama, Japan, [email protected] 2: Department of System Design Engineering, Yokohama, Japan, [email protected]

3: Department of System Design Engineering, Yokohama, Japan, yohei@ sd.keio.ac.jp

Abstract A novel velocity measurement technique was proposed for investigation of nanoscale flow structure in the vicinity of electrolyte-glass interface. The technique enables the velocity measurement of the ion existing on the order of tens of nanometers from a microchannel wall due to the caged fluorescent dye and evanescent wave illumination. Caged fluorescent dye is initially non-fluorescent because a chemical group is attached to quench the fluorescence of the dye. After irradiated by ultraviolet light, the chemical group is cleaved and the dye restored the fluorescence. In order to excite the uncaged dye which exists in the vicinity of the interface selectively, evanescent wave illumination is examined. Evanesent wave penetrates into the electrolyte when total internal reflection occurred on the interface. The velocity of the uncaged dye was obtained by tracing fluorescence from the uncaged fluorescent dye. A comparison between the fluid flow measured by using evanescent wave and epi-fluorescent illumination was investigated. The molecular tagging technique with two types of illumination was applied to a pressure-driven flow and an electroosmotic flow (EOF) in an I-shaped microchannel with the 50 μm × 400 μm rectangular cross-section. The microchannel was comprised of a poly(dimethlsiloxane) (PDMS) chip and a ∅50 mm diameter 170 μm thickness borosilicate cover glass. The working fluid was prepared by dissolving CMNB-caged fluorescein and DMNB-caged fluorescein dextran into a carbonate buffer at pH 9.3. A pulsed ultraviolet laser beam was focused as a sheet into the microchannel to cleave the chemical group of the caged dye. The uncaged fluorescent dye was excited by epi-fluorescent illumination using a mercury lamp or evanescent wave illumination. For evanescent wave illumination, a 60× magnification objective lens with a numerical aperture of 1.45 was utilized to generate evanescent wave. The measured velocity of pressure-driven flow by evanescent wave illumination showed smaller than that by epi-fluorescent illumination, while the velocity difference between both illumination was not observed in EOF. The present study exhibited that the evanescent wave molecular tagging technique has the ability to investigate the nano-scale flow structure in the vicinity of the electrolyte-glass interface. 1. Introduction Development of micro- and nanotechnology will contribute to great advance of microminiatureized chemical analysis system whose processes, e.g. mixing, reaction, separation and so forth, are integrated on a small chip comprised of microchannels with a few hundred μm width. The small liquid sample volumes in a microchannel are handled by a force on the liquid, which is generally exerted by an external electric field. This transport mechanism is well known as electromomotic flow (EOF), in which the motion of ions is induced. The EOF structure is governed by the formation of positive ions formed close to the solid surface of a microchannel known as electric double layer (EDL), because the electromigration of ions in the EDL induces the ion drag on the rest of liquid in a microchannel. Therefore, quantitative measurements of velocity of the ions that exist on the order of nano meters from channel wall will contribute to the accurate control of transport phenomena affected by EDL, which is the important issue of development of the microminiaturized and integrated system. To date, several novel measurement techniques for flow velocity in a microchannel have been developed. An application of particle image velocimetry (PIV) to microscale flow was proposed as micro-PIV, which can measure the flow velocity with a microscale resolution by using submicron


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particles (Santiago et al. 1998). For a nanoscale resolution near the microchannel wall, nano-particle image velocimetry (nano-PIV) realizes a near-wall velocity measurement for EOF (Sadr et al. 2004). In the velocity measurement using submicron particles, the influence of the lift force acting on a particle has been vaguely discussed. A dye-based measurement technique, i.e., a molecular tagging technique using caged fluorescent dye has been developed to measure the fluid velocity in both macro- and microscale. First experimental efforts using caged fluorescent dye is conducted in macroscale flow (Lempert et al. 1995). As the application to microscale flow, a few previous works have reported the velocity distribution of a pressure-driven flow and an EOF in capillaries and microchannels (Paul et al. 1998, Ross et al. 2001, Sinton and Li 2003). However, it is difficult to detect the flow structure in the vicinity of the electrolyte-glass interface, especially, electrokinetic effects on velocity field, because their measurements remain restricted to a spatial resolution on the order of a few μm. The present study proposes the measurement technique for velocity of ions, which enables to detect ions selectively in the vicinity of electrolyte-glass interface, termed as evanescent wave molecular tagging (EWMT) technique. Caged fluorescent dye is initially non-fluorescent because a chemical group is attached to quench the fluorescence of the dye. After irradiated by ultraviolet light, the chemical group is cleaved and the dye restored fluorescence. In order to excite the uncaged dye which exists in the vicinity of the interface, evanescent wave illumination is employed. The evanesent wave penetrates into the electrolyte when total internal reflection occurred at the interface. The velocity of the uncaged dye was obtained by tracing fluorescence from the uncaged fluorescent dye. A comparison between the fluid flow measured by using evanescent wave and epi-fluorescent illumination was investigated. 2. Measurement technique 2.1 Evanescent wave illumination Total internal reflection occurs when a laser beam propagating through transparent medium of high-refractive index reaches on an interface with one of low-refractive index with higher incidence angle than critical angle (Axelrod et al. 1984), as illustrated in Fig 1, which defined as:

θc = sin−1 n2

n1

, (1) where θc is the critical angle, n1 and n2 are the refractive indices of the first medium and the second medium, respectively. While the beam is totally internally reflected at the interface, evanescent wave, which penetrates into the medium of lower refractive index, is emerged. The evanescent wave intensity, Ieva, decays exponentially with the distance, z, from the interface, given by:

Ieva (z ) = Ieva (0)exp − z

z p

⎛

⎝ ⎜ ⎜

⎞

⎠⎟⎟, (2)

ZEvanescent wave

Laser

Electric double layer

Z

Ieva0 I0I0 /e

zp

n1

n2

θ > θc

ZEvanescent wave

Laser

Electric double layer

Z

Ieva0 I0I0 /e

zp

n1

n2

θ > θc

(a) (b)

Fig. 1. Schematic of (a) total internal reflection and (b) the evanescent wave intensity

13th Int Symp on Applications of Laser Techniques to Fluid Mechanics Lisbon, Portugal, 26-29 June, 2006 #1334

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where Ieva(0) is the evanescent wave intensity at the interface and zp is the characteristic penetration depth of he evanescent wave, defined as follows:

z p = λ

4π n12sin2θi − n2

2, (3)

where, λ is the laser beam wavelength in vacuum. Therefore, evanescent wave illumination can excite selectively fluorescent dye at the interface region on the order of the laser beam wavelength (from tens nanometers to hundreds nanometers). 2.2 Principle of evanescent wave molecular tagging technique using caged fluorescent dye Caged fluorescent dye is initially non-fluorescent because a chemical group is attached to quench the fluorescence of the dye. After irradiated by ultraviolet light, the chemical group is cleaved and the dye restored the fluorescence, known as an uncaging event. This is irreversible and requires on the order of milliseconds at least (Lempert et al. 1995). Figure 2(a) illustrates the schematic of the molecular tagging technique with epi-fluorescent illumination. Just after the pulsed ultraviolet laser beam induces an uncaging event for the dye, the uncaged fluorescent dye is excited by an illumination light, e.g., a mercury lamp. Evanescent wave molecular tagging technique utilizes the evanescent wave, as exhibited in Figure 2(b). The evanescent wave enables us to detect the fluorescence from the dye that exists in the vicinity of wall surface. The dye is diffused by the flow convection. The instantaneous fluorescence intensity profile is fitted to Gaussian profile (Yamamoto et al. 2002). The position of the fluorescent intensity peak-value is obtained by Gauss fitting. The observed dye velocity is calculated from the peak-value displacement and the interval of the images.

Flow direction

~ 1

mm

Z X

Pulsed ultraviolet laser beam(a)Wall

Illuminated region

Flow direction

Wall

few hundreds nanometersZ X

Pulsed ultraviolet laser beam

Illuminated region

(b)

Fig. 2. Schematic concept of (a) epi-fluorescent illumination and (b) evanescent wave illumination.

Displacement of uncagedfluorescent dye

Flow direction

Flow direction

uncaged fluorescent dye

Y X

Y X

xΔ

0tt =

ttt Δ+= 0

0tt =xΔ

X

0tt =

ttt Δ+= 0

Fluo

resc

ent

inte

nsity

X

Fluo

resc

ent

inte

nsity

(a)

(b)

Fig. 3. Schematic concept of the calculation of the dye velocity.


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3. Experimental setup 3.1 Caged fluorescent dye The DMNB-caged fluorescein dextran (Molecular Probes Inc.) and CMNB-caged fluorescein (Molecular Probes Inc.) were used as the caged fluorescent dye. The properties of the dyes are compiled in table 1. The DMNB-caged fluorescent dye is selected for the pressure-driven flow because of redusing diffusion due to large molecular weight (MW = 8000 - 12000), however, Paul et al. (1998) reported that the undesired separation effect was observed due to its multicomponent nature. Thus, the CMNB-caged fluorescein as a single component dye is employed for the EOF. The DMNB-caged fluorescein dextran and CMNB-caged fluorescein was prepared by dissolving with a 20 μmol/l and 0.2 mmol/l concentration, respectively, in 10 mmol/l carbonate buffer at pH 9.3. 3.2 Microchannel An I-shaped microchannel shown in Fig. 4 was comprised of a poly(dimethylsiloxane) (PDMS) chip which was fabricated by soft lithography and a ∅50mm diameter 170 μm thickness borosilicate cover glass plate (Matsunami glass ind., LTD. ). Cylindrical reservoirs were attached on the PDMS at the end of the microchannel. The pressure-driven flow was generated by the water head between two reservoirs. For the EOF, a DC electric field 34.5 V/cm was applied with platinum electrodes immersed in the reservoirs. 3.3 Imaging apparatus A schematic of the present measurement system is illustrated in Fig. 5. A Peltier-cooled CCD camera (Hamamatsu Photonics, K.K., C4880-80) with a 656 × 494 array was mounted on an inverted microscope (Nikon Corp., TE2000U). A continuous ultraviolet laser beam from an Nd:YVO4 laser (DPSS Inc., 3510−30) operating at a wavelength of 355 nm and a power of 1 W was used for the uncaging event. The beam, whose power was controlled to be 0.36 W by an attenuator , was pulsed by a shutter (Vincent Associate, VMM-T1) in 10 ms pulse duration. The pulsed beam was focused with a plano-convex cylindrical lens (f = 50 mm) into a laser sheet in a microchannel. In order to generate evanescent wave illumination, a 60× magnification oil-immersion objective lens (Nikon Corp., CFI Plan Apo. TIRF) with a numerical aperture of 1.45 was employed as illustrated in Fig. 6. For epi-fluorescent illumination, the 60× magnification or a 20× magnification (Nikon Corp., CFI S Fluor) was employed. The measurement region is illustrated in Fig. 7. The objective lens was focused on the XY plane of z = 0 μm and z = 25 μm for evanescent wave and epi-fluorescent illumination, respectively.

Table 1. Prperties of caged fluorescent dye CMNB-caged

fluorescein DMNB-caged

fluorescein dextran Molecular weight 826.81 10000 Absorption wavelength* [nm] 333 353 Absorption wavelength [nm] 494 494 Emission wavelength [nm] 518 518 (Appproximate maximum values) * For uncaging event


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XY

400 μmInlet Outlet

29 mm

400 μm

50μm

170

μm

YZ

PDMS

Borosilicate glass cover slip

(a)

(b)

Mirror Mirror

MirrorLens

Lens

Mercury lamp

Filter block

Filter block

Cooled CCD camera Laser (473nm, 30mW)

Beam expanderLaser (355nm, 1W)

Attenuator

Shutter

MicrochannelMicroscope stage

Objective lens XY

0.6× TV lens

Fig. 4. Schematic illustration of the microchannel in (a) top view and (b)cross-sectional view.

Fig. 5. Schematic illustration of imaging apparatus.

MicrochannelLaser beam (355nm)

Laser beam (473nm)

Objective lens (60 ×, N.A. = 1.45)

Cover glass

Back focal plane

Microcscope stage

Microchannel

Measurement region by 60× objective lens

Measurement region by 20× objective lens

XY

400 μm

Fig. 6. Schematic illustration of the total internal microscope using a 60× magnification objective lens.

Fig. 7. Schematic illustration of measurement region.

A laser beam from a diode pumped solid laser (Laser-Compact Co.Ltd, LCS-DTL-364)

operating at a wavelength of 473 nm and a power of 30 mW was used to generate evanescent wave and excite the uncaged fluorescent dye. The beam was collimated and expanded by a beam expander, comprised of a 10× magnification objective lens, a pinhole (d = 20 μm) and a plano-convex lens (f = 500 mm). The expanded beam was focused on the back focal plane at the peripherals region of the 60× magnification objective lens, passing through a plano-convex lens (f = 600 mm), a plane mirror and a dichroic mirror. The total internal reflection angle was adjusted to be 70° by the X-direction translation of the plane mirror, which resulted in 74 nm of the characteristic penetration depth. The collimated laser beam from the objective lens passed through the immersion oil and total internal reflection occurred on an interface between the borosilicate cover glass of 170 μm thickness and the buffer solution. The present system can be easily switched to epi-fluorescent illumination using a mercury lamp by changing the optical path in the microscope. Fluorescence from the uncaged dye was captured onto the CCD pixel sheet through the objective lens, an emission filter transmitting wave length longer than 520 nm and 0.6× magnification TV lens. The size of the CCD pixel sheet along with the objective lens corresponded


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to a measurement area of 180 μm × 136 μm for the 60× objective lens and 408 μm × 541 μm for the 20× objective lens in the object plane. A frame grabber (Coreco Inc., IC-PIC) captured time-series images with 37 ms frame interval. For pixel error reduction, an average per 5 × 5 pixels was performed. In the present experiment, a spatial resolution of molecular tagging technique was 1.4 μm in the Y-direction for the 60× objective lens and 4.1 μm in Y-direction for the 20× objective lens. As the captured images were depended on the excitation light intensity and background noise of the CCD camera, a postprocessing was conducted to correct the fluorescent intensity. The luminous intensity of the image captured without the excitation light. The background noise was subtracted from the detected intensity. After the subtraction, the intensity was normalized by the fluorescent intensity of images captured under the excitation light with the long exposure time where small amount of uncaged dye homogeneously dispersed in the aqueous solution. 4. Results and discussion 4.1 Molecular tagging technique with epi-fluorescent illumination For the investigation of the bulk flow structure in the microchannel, dye velocities of the pressure-driven flow and the EOF were measured by using 20× objective lens and epi-fluorescent illumination. Figure 8 shows the time-evolution of instantaneous image of uncaged fluorescence in the pressure-driven flow. The fluorescent intensity at y = 200 μm in Fig. 8 is plotted in Fig. 9. The fluorescent intensity profile broadened with migration to the X-direction keeping Gaussian shape. The position of the peak-value of fluorescent intensity profile was obtained by Gaussian fitting. The fluorescent intensity profiles of 51 images in time series resulted in 50 velocities at each Y-direction positions. The RMS value of the velocity at y = 200 μm was estimated to be ± 15.1 μm/s (16.8 % of the velocity). This error could be derived from Gaussian fitting error caused by undesired fluorescent intensity profiles and the fluctuation of the CCD camera frame interval whose RMS value was estimated to be ± 0.52 ms (1.42% of the averaged frame interval). The averaged velocity at each Y-position is plotted in Fig. 10. The trapezoidal shape of the velocity profile agrees with the theoretical result (Brody et al. 1996). This result shows that the velocity profile has uniform velocity region at the center of the microchannel (at y = 100 - 300 μm) in terms of the Y-direction. In the EOF, the time-evolution of instantaneous image of uncaged fluorescence is shown in Fig. 11. The fluorescent intensity at y = 200 μm and the calculated velocity profile from 51 images are shown in Fig. 12 and Fig. 13, respectively. The RMS value of the velocity at y = 200 μm was estimated to be ± 6.46 μm/s (4.67 % of the velocity). The velocity profile of the EOF in Fig. 13 has a flow pattern that superimposed a reverse-trapezoidal and a plug flow pattern. When the external electric field is applied, the electromigration of positive ions in the EDL exerted the bulk solution flow in the microchannel because the flow is governed by viscosity. It is shown theoretically that the flow shows a plug flow pattern (Probstein, 1994) in the EOF. Taylor and Yeung (1993) and Sato et al. (2002) reported that a reverse-parabolic flow pattern was observed at a depth-wise midpoint of a microchannel. They measured the eloctroosmotic flow velocity with the pressure-driven flow which was induced by elevating the water head in the cathode-side inlet due to the electroosmotic flow. The result in the present experiment has good agreement with the flow pattern reported in the previous experimental works.


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0 100 200 300 400 500

0.0

0.5

1.0

1.5

2.0

2.5Col 8 vs Col 9 Col 8 vs Col 10 Col 8 vs Col 11 Col 8 vs Col 12

Fluo

resc

ence

inte

nsity

[-]

X-direction position [μm]

0 ms370 ms740 ms1110 ms

0 100 200 300 400

0

20

40

60

80

100

Y-direction position [μm]

Dye

vel

ocity

[μm

/s]

Fig. 9. Fluorescence intensity profiles at y = 200 μm in the pressure-driven flow with epi-fluorescent illumination.

Fig. 10. The calculated velocity profile in the pressure-driven flow.

Y

X

408 μm

541 μm

(a) (b) (c) (d)

Fig. 8. Time-evolution of instantaneous image of uncaged fluorescence in the pressure-driven flow at (a) 0 ms, (b) 370 ms, (c) 740 ms and (d) 1110 ms.

Y

X

408 μm

541 μm

(a) (b) (c) (d)

Fig. 11. Time-evolution of instantaneous image of uncaged fluorescence in the EOF at (a) 0 ms, (b) 370 ms, (c) 740 ms and (d) 1110 ms.


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0 100 200 300 400 500

0.00.51.01.52.02.53.0

Col 6 vs Col 7 Col 6 vs Col 8 Col 6 vs Col 9 Col 6 vs Col 10

0 ms370 ms740 ms1110 ms

Fluo

resc

ence

inte

nsity

[-]

X-direction position [μm] 0 100 200 300 400

020406080

100120140160


Dye

vel

ocity

[μm

/s]

Fig. 12. Fluorescence intensity profiles at y = 200 μm in the EOF with epi-fluorescent illumination.

Fig. 13. The calculated velocity profile in the EOF.

4.2 Molecular tagging technique with evanescent wave illumination

Figures 14 and 15 exhibit the time-evolution of instantaneous image of uncaged fluorescence in the pressure-driven flow with epi-fluorescent and evanescent wave illumination captured by the 60× objective lens, while the fluorescence intensity profiles at y = 200 μm of Fig. 15 is plotted in Fig. 16. The velocity profile calculated from 21 images is depicted in Fig. 17. The RMS value of the velocity at y = 200 μm was estimated to be ± 4.52 (6.14 % of the velocity) μm/s for epi-fluorescent illumination and ± 9.89 μm/s (37.4 % of the velocity) for evanescent wave illumination, respectively. The both of the velocity profiles have a flat pattern because the measurement region is the center of the microchannel (y = 136 – 272 μm) where the uniform velocity patterns at y = 100 – 300 μm present in the both flows as verified in the section 4.1. It is obvious that the velocity obtained by evanescent wave illumination was much smaller than that by epi-fluorescent illumination. The velocity profile in the Z-direction in the present microchannel is predicted theoretically to form a parabolic pattern (Brody et al. 1996) and the velocity near z = 0 μm in the pressure driven flow is expected to be much decreasing compared to the bulk velocity because of the non-slip condition at wall; Therefore, the result indicates the present technique can detect the velocity in the near wall region.

Measurements of the dye velocity in the EOF were carried out in order to investigate the electrokinetic effect by the EDL on the velocity field. Figures 18 and 19 show the time-evolution of instantaneous image of uncaged fluorescence in the EOF with epi-fluorescent illumination and evanescent wave illumination captured by the 60× objective lens. Figure 20 exhibits the fluorescence intensity profiles at y = 200 μm in Fig. 19. The calculated velocity profile was shown in Fig. 20. The RMS value of the velocity at y = 200 μm was estimated to be ± 7.56 μm/s (7.11 % of the velocity) for epi-fluorescent illumination and ± 8.60 μm/s (6.95 % of the velocity) for evanescent wave illumination, respectively. It is expected that the large velocity gradient in the Z-direction exists in the EDL to satisfy the non-slip condition at the wall; however, the present experiment cannot detect an evidence of the velocity gradient, i.e., the significant differences of the dye velocity between the two illumination methods. It is considered that the observed velocity by EWMT technique is affected by the magnitude between thickness of the EDL in the buffer solution and the characteristic penetration depth of the evanescent wave. The Debye length well known as EDL thickness is given by:


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21

0222 ⎟⎟

⎠

⎞⎜⎜⎝

⎛=

czFRT

Dελ , (4)

where ε is the permittivity of liquid, R is the gas constant, T is temperature, F is the Faraday constant, z is the charge number of ion and c0 is the bulk ion concentration. The Debye length in the 10 mmol/l carbonate buffer is estimated as to be 3 nm. This value is much smaller than the characteristic penetration depth of the evanescent wave in the present experiment, i.e., 74 nm. Therefore, the difference was not observed between epi-fluorescent and evanescent wave illumination in the EOF since this estimation indicates that the most of illuminated uncaged dye was expected to exist outside of the EDL. In the future work, further investigation of the flow structure in the vicinity of the interface will be carried out by adjusting the ion concentration of the buffer solution to set the EDL thickness to be the equivalent to the characteristic penetration depth of evanescent wave illumination.

Y

X

136 μm

180 μm

(a) (b) (c) (d)

Fig. 14. Fluorescence images of the uncaged dye in the pressure-driven flow captured by the ×60 objective lens with epi-fluorescent illumination at (a) 0 ms, (b) 185 ms, (c) 370 ms and (d) 555 ms.

Y

X

136 μm

180 μm

(a) (b) (c) (d)

Fig. 15. Fluorescence images of the uncaged dye in the pressure-driven flow captured by the ×60 objective lens with evanescent wave illumination at (a) 0 ms, (b) 370 ms, (c) 740 ms and (d) 1110 ms.


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0 20 40 60 80 100 120 140 160 1800.00.51.01.52.02.53.03.5

Col 8 vs Col 10 Col 8 vs Col 11 Col 8 vs Col 12

0 ms370 ms740 ms1110 ms

Fluo

resc

ence

inte

nsity

[-]

X-direction position [μm]140 160 180 200 220 240 260

0

20

40

60

80

100

120Epi-fluorescent illuminationEvanescent wave illumination


Dye

vel

ocity

[μm

/s]

Fig. 16. Fluorescence intensity profiles at y = 200 μm in the pressure-driven flow with evanescent wave illumination.

Fig. 17. Calculated velocity profile in the pressure-driven flow.

Y

X

136 μm

180 μm

(a) (b) (c) (d)

Fig. 19. Fluorescence images of the uncaged dye in the EOF captured by the ×60 objective lens with evanescent wave illumination at (a) 0 ms, (b) 185 ms, (c) 370 ms and (d) 555 ms.

Y

X

136 μm

180 μm

(a) (b) (c) (d)

Fig. 18. Fluorescence images of the uncaged dye in the EOF captured by the ×60 objective lens with epi-fluorescent illumination at (a) 0 ms, (b) 185 ms, (c) 370 ms and (d) 555 ms.


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0 20 40 60 80 100 120 140 160 180

0

2

4

6

8Col 5 vs Col 6 Col 5 vs Col 7 Col 5 vs Col 8 Col 5 vs Col 9

0 ms

370 ms

Fluo

resc

ence

inte

nsity

[-]

X-direction position [μm]

185 ms

555 ms

140 160 180 200 220 240 2600

20406080

100120140

Epi-fluorescent illuminationEvanescent wave illumination


Dye

vel

ocity

[μm

/s]

Fig. 20. Fluorescence intensity profiles at y = 200 μm in the EOF with evanescent wave illumination.

Fig. 21. Calculated velocity profile in the EOF.

5. Conclusion A velocity measurement technique in the vicinity of the electrolyte-glass interface, utilizing the caged fluorescent dye and evanescent wave illumination, was developed in order to investigate flow structure affected by the electric double layer. The important conclusions in the present study are summarized bellow. (1) The velocity profiles in a microchannel were obtained by a molecular tagging techniques using epi-fluorescent and evanescent wave illumination. The techniques were applied to the pressure-driven flow and the EOF. The spatial resolution of the measurement was 1.4 μm in the Y-direction for the 60× objective lens and 4.1 μm in the Y-direction for the 20× objective lens, respectively. (2) The evanescent wave molecular tagging technique was performed compared to the technique using epi-fluorescent illumination. The velocity near the wall region was decreasing compared to the bulk flow in the case of the pressure-driven flow. In the EOF, the difference of the velocity between the two illumination methods was not observed, because the magnitude between the thickness of the EDL and the characteristic penetration depth of the evanescent wave affects the velocity field measured by the EWMT technique. Acknowledgement This work was subsidized by the Grant-in-Aid for Young Scientists (A) No.17686017 of Ministry of Education, Culture, Sports, Science and Technology and TEPCO Research Foundation. Reference Axelrod, D., Burghardt, T.P. and Thompson, N.L. (1984). “Total Internal Reflection Fluorescence,” Annual review of biophysics and bioengineering, 13, pp. 247−268. Brody, J.P., Yager, P., Goldstein, R.E. and Austin, R.H. (1996) “Biotechnology at Low


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Reynolds Numbers,” Biophysical Journal, 71, pp. 3430−3441. Lempert, W.R., Magee, K., Ronney, P., Gee, K.R. and Haugland, R.P. (1995). “Flow tagging velocimetry in incompressible flow using photo-activated nonintrusive tracking of molecular motion (PHANTOMM),” Experiments in Fluids, 18, pp. 249−257. Paul, P.H., Garfuilo, M.G. and Rakestraw, D.J. (1998). “Imaging of pressure and electrokinetically driven flows through open capillaries,” Analytical Chemistry, 70, pp. 2459−2467. Probstein, R.F. (1994). Physicochemical Hydrodynamics: An Introduction Second Edition, A wiley-Interscience Publication. Ross, D., Johnson, T.J. and Locascio, L.E. (2001). “Imaging of electroosmotic flow in Plastic microchannels,” Analytical Chemistry, 73, pp. 2509−2515. Sadr, R., Yoda, M., Zheng, Z. and Conlisk, A.T. (2004). “An experimental study of electro-osmotic flow in rectangular microchannels,” Journal of Fluid Mechanics, 506, pp. 357−367. Santiago, J.G., Wereley, S.T., Meinhart, C.D., Beebe, D.J. and Adrian, R.J. (1998). “A particle image velocimetry system for microfluidics,” Experimets in Fluids, 25, pp. 316−319. Sato, Y., Hishida, K. and Maeda, M. (2002) “Quantitative Measurement and Control of Electrokinetically Driven Flow in Microspace,” Micro Total Analysis Systems 2002, 1, pp. 512−514. Sinton, D.A. and Li, D. (2003). ”Microfluidic velocimetry with near-wall resolution,” International Journal of Thermal Sciences, 42, pp. 847−855. Taylor, A. and Yeung, ES. (1993). “Imaging of Hydrodynamic and Electrokinetic Flow Profiles in Capillaries,” Analytical Chemistry, 65, pp. 2928−2932. Yamamoto, T., Inaba, S., Sato, Y., Hishida, K., and Maeda, M. (2002). “Measurements in microchannel by laser induced molecular tagging and micro-PIV,” 11th International Symposium on Application of Laser Techniques to Fluid Mechanics: Web-site.

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