Laser-induced mixing in microfluidic channels

Article abstract of DOI:10.1021/ac070081i

We demonstrate a novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems. This method utilizes highly focused nanosecond laser pulses from a Q-switched Nd:YAG laser at λ = 532 nm to generate cavitation bubbles within 100- and 200-μm-wide microfluidic channels containing the parallel laminar flow of two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. We use time-resolved imaging and fluorescence detection methods to visualize the mixing process and to estimate both the volume of mixed fluid and the time scale for the re-establishment of laminar flow. The results show that mixing is initiated by liquid jets that form upon cavitation bubble collapse and occurs ～20 μs following the delivery of the laser pulse. The images also reveal that mixing occurs on the millisecond time scale and that laminar flow is re-established on a 50-ms time scale. This process results in a locally mixed fluid volume in the range of 0.5-1.5 nL that is convected downstream with the main flow in the microchannel. We demonstrate the use of this mixing technique by initiating the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to yield fluorescent resorufin. This approach to generate the mixing of adjacent fluids may prove advantageous in many microfluidic applications as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation.

Full text of DOI:10.1021/ac070081i

Anal. Chem. 2007, 79, 4484-4492
Laser-Induced Mixing in Microfluidic Channels
Amy N. Hellman,†,‡,§ Kaustubh R. Rau,
†,‡,|,⊥
Helen H. Yoon, Stephanie Bae, James F. Palmer,
#
†
¶
¶
¶,#
,†,‡,|
K. Scott Phillips, Nancy L. Allbritton, and Vasan Venugopalan*
Department of Biomedical Engineering, Department of Chemical Engineering and Materials Science, Department of
Chemistry, Laser Microbeam and Medical Program, Beckman Laser Institute, and Department of Physiology and Biophysics,
University of California, Irvine, Irvine, California 92697, and Department of Bioengineering, University of California,
San Diego, La Jolla, California 92093
community.1-7 Microfluidic devices possess characteristic channel
widths in the range W ∼ 50-500 µm with volumetric flow rates
of Q ∼ 50-500 µL/h resulting in Reynolds numbers of Re , 10.
The resulting laminar flow admits only diffusion as a mechanism
to mix two adjacent fluid streams. The reliance on diffusion alone
for the mixing of reactants results in extremely long mixing times
and lengths. Specifically, molecular diffusion across a distance of
We demonstrate a novel strategy for mixing solutions and
initiating chemical reactions in microfluidic systems. This
method utilizes highly focused nanosecond laser pulses
from a Q-switched Nd:YAG laser at λ ) 532 nm to
generate cavitation bubbles within 100- and 200-µm-wide
microfluidic channels containing the parallel laminar flow
of two fluids. The bubble expansion and subsequent
collapse within the channel disrupts the laminar flow of
the parallel fluid streams and produces a localized region
of mixed fluid. We use time-resolved imaging and fluo-
rescence detection methods to visualize the mixing pro-
cess and to estimate both the volume of mixed fluid and
the time scale for the re-establishment of laminar flow.
The results show that mixing is initiated by liquid jets that
form upon cavitation bubble collapse and occurs ∼20 µs
following the delivery of the laser pulse. The images also
reveal that mixing occurs on the millisecond time scale
and that laminar flow is re-established on a 50-ms time
scale. This process results in a locally mixed fluid volume
in the range of 0.5-1.5 nL that is convected downstream
with the main flow in the microchannel. We demonstrate
the use of this mixing technique by initiating the horse-
radish peroxidase-catalyzed reaction between hydrogen
peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphe-
noxazine (Amplex Red) to yield fluorescent resorufin. This
approach to generate the mixing of adjacent fluids may
prove advantageous in many microfluidic applications as
it requires neither tailored channel geometries nor the
fabrication of specialized on-chip instrumentation.
1
00 µm requires times of 10-1000 s when considering molecules
-3
-5
2
8
with diffusivities in the range of 10 -10 mm /s. This translates
into mixing lengths of 0.1-10 m. Such mixing times and lengths
are clearly impractical for rapid mixing of small volumes. Thus
microfluidic devices that aim to provide rapid kinetic biochemical
analysis cannot rely on diffusion alone to mix reagents. Several
articles have considered in detail these issues pertaining to flow
and transport at the microscale.8
-11
The limitations posed by diffusion-based transport have spurred
researchers to develop various strategies to rapidly mix small
volumes in microfluidic devices. Generally these strategies either
employ changes in channel geometry (static mixers) or introduce
external energy sources (active mixers) to enhance fluid contact
or destabilize the laminar flow.12 In static mixers, the simplest
design modifications are to increase channel length and tortuosity
to increase fluid contact time and enhance diffusion. The use of
hydrodynamic focusing to achieve reductions in both the flow
cross section and molecular diffusion time has also been
13-15
described.
Such hydrodynamic focusing strategies have
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1
(
(
4
7
The challenge of rapidly mixing two fluids within a laminar
flow has received considerable attention within the microfluidics
(
(
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*
To whom correspondence should be addressed. Phone: (949) 824-5802.
(7) Gartecki, P.; Fuerstman, M. J.; Fischbach, M. A.; Sia, S. K.; Whitesides, G.
Fax: (949) 824-2541. E-mail: vvenugop@uci.edu.
M. Lab Chip 2006, 6, 207-212.
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(9) Squires, T. M.; Quake, S. R. Rev. Mod. Phys. 2005, 77, 977-1026.
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73, 101-108.
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†
Department of Chemical Engineering and Materials Science, University of
California, Irvine.
‡
Laser Microbeam and Medical Program, Beckman Laser Institute, University
of California, Irvine.
§
Department of Bioengineering, University of California, San Diego.
Department of Biomedical Engineering, University of California, Irvine.
Current address: National Centre for Biological Sciences, Tata Institute of
|
⊥
Fundamental Research, Bangalore 560 065, India.
#
Department of Chemistry, University of California, Irvine.
Department of Physiology and Biophysics, University of California, Irvine.
(13) Knight, J. B.; Vishwanath, A.; Brody, J. P.; Austin, R. H. Phys. Rev. Lett.
1998, 80, 3863-3866.
¶
4484 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
10.1021/ac070081i CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/18/2007

provided mixing times as rapid as 8 µs.16 In other areas,
researchers have designed static mixers that utilize chaotic
advection, a process in which fluid interfaces undergo stretching
and folding to achieve higher mixing efficiencies. Mixers that
employ chaotic advection can involve complex designs with three-
dimensional microchannels or relief features on the microchannel
17-19
floor to achieve mixing of two fluid streams.
In active mixers,
an energy source is used to destabilize the laminar flow. A simple
case of this approach utilizes the temporal pulsing of the flow rate
of two fluid streams and has been shown to create well-mixed
regions within 250 ms in a 200 µm × 120 µm channel at a flow
2
0
21,22
rate of 1.55 µL/s. Designs employing ultrasound,
electroos-
2
3
24
25,26
motic flow, dielectrophoresis, and magnetic microstirrers
have all been examined to mix two fluids within microchannels.
Thus, a multitude of approaches have been proposed to handle
the microfluidic mixing problem. The choice of mixer type is best
determined by the specific application and other considerations
such as complexity of design and fabrication.
In this report, we present a new method to provide rapid,
localized mixing of fluids in microfluidic channels on demand. The
concept utilizes the delivery of a highly focused nanosecond laser
pulse resulting in plasma formation and subsequent cavitation
bubble generation. The three-dimensional character of the fluid
flow associated with the cavitation dynamics causes two adjacent
fluid streams to mix on microsecond to millisecond time scales.
This laser-based method of fluid mixing has several important
characteristics that distinguish it from those using more conven-
tional active mixing methods such as ultrasound. First, laser-
generated cavitation produces a volume of mixed fluid only in the
region surrounding the laser focal volume, thereby enabling the
production of a locally mixed fluid volume within the microfluidic
channel. Second, the focal volume of the laser beam can be moved
easily, and thus, the site of mixing is not fixed at a particular
location in the device. Third, the laser delivery can be easily
switched on and off to provide fluid mixing only at specific times
in a microfluidic process. Last, since the laser is decoupled from
the microfluidic device, this approach provides microfluidic mixing
capabilities without requiring any modification to the microfluidic
device design or fabrication. Pulsed laser microbeams also offer
Figure 1. Setup for laser-induced mixing and time-resolved imaging.
fluorescence excitation, and uncaging of photoexcitable com-
pounds.28 Finally, one can envision new applications wherein the
use of an on-demand, localized mixing capability could prove
useful, for example, to mix small volumes of two reactants to
synthesize small quantities of product.
In this study, a highly focused Q-switched Nd:YAG laser
emitting 6-ns duration pulses at λ ) 532 nm was used to produce
mixing within the parallel flow of two adjacent fluids within a
microfluidic channel. The dynamics and extent of mixing were
assessed in three studies. First, time-resolved photography was
used to visualize the spatiotemporal dynamics of the cavitation-
induced mixing at the site of laser pulse delivery on nanosecond
to millisecond time scales. Second, a fluorescence detection
system was used to examine the spatial extent of the mixed bolus
of fluid ∼7 mm downstream from the site of pulsed laser delivery.
Third, fluorescence video microscopy was used to visualize the
efficacy of this mixing approach to initiate a biochemical reaction.
This was done by establishing the parallel flow of two adjacent
fluid streams containing nonfluorescent substrate and catalyst
molecules that, when mixed, yield a fluorescent product.
additional capabilities in a microfluidic context and can provide a
multifunctional tool for capabilities such as selective cell lysis,27
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MATERIALS AND METHODS
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Whitesides, G. M. Science 2002, 295, 647-651.
Microfluidic Device. The microfluidic device was fabricated
from poly(dimethylsiloxane) (PDMS) using casting techniques.
A SU-8 photoresist layer was spin coated onto a silicon wafer, and
the desired pattern was etched using standard photolithography
techniques. The channel design (shown in the lower left-hand
corner of Figure 1) consisted of two inlets and an outlet in the
form of a Y, with channel widths of either 100 or 200 µm and a
channel depth of 50 µm. Two-part silicone resin (Sylgard 184, Dow
Corning Corp.) was mixed in a 10:1 ratio (part A/part B, v/v),
poured over the silicon master, degassed, and cured at 70 °C for
(
(
(
(
20) Glasgow, I.; Aubry, N. Lab Chip 2003, 3, 114-120.
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2002, 2, 151-157.
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(
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22) Yaralioglu, G. G.; Wygant, I. O.; Marentis, T. C.; Khuri-Yakub, B. T. Anal.
Chem. 2004, 76, 3694-3698.
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832.
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6-39.
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(
(
25) Lu, L. H.; Ryu, K. S.; Liu, C. J. Microelectromech. Syst. 2002, 11, 462-469.
26) Suzuki, H.; Kasagi, N.; Ho, C. M. Proc. 3rd Int. Symp. Turbulence Shear
Flow Phenomema 2003, 817-822.
1
h. The cured polymer was peeled from the silicon wafer and
(
27) Krasieva, T. B.; Chapman, C. F.; Lamorte, V. J.; Venugopalan, V.; Berns,
(28) Paul, P. H.; Garguilo, M. G.; Rakestraw, D. J. Anal. Chem. 1998, 70, 2459-
M. W.; Tromberg, B. J. Proc. SPIE 1998, 3260, 38-44.
2467.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4485

sealed against a glass coverslip after punching inlet and outlet
vias for fluid access.
Time-Resolved Bright-Field Imaging of Laser-Induced
Mixing. Figure 1 is a schematic of the experimental system used
in the time-resolved imaging experiments. Fluid was metered into
the microfluidic channels using two 100-µL syringes (Gastight No.
8
1026, Hamilton Co.) that were connected to the device using
Teflon tubing and driven by a syringe pump (Pump 11, Harvard
Apparatus). Two fluid streams consisting of water and 8 mg/mL
Naphthol Green dye (Sigma), respectively, were supplied from
separate syringes each at a flow rate of 50 µL/h into individual
channels of 100- or 200-µm width. The total flow rate of 100 µL/h
resulted in mean flow velocities of 2.8 and 5.6 mm/s in channels
of 200- and 100-µm width, respectively.
The microfluidic device was placed on the sample stage of an
inverted microscope (Zeiss Axiovert A110), and the λ ) 532 nm
output from a frequency-doubled Q-switched Nd:YAG laser (INDI
I-10, Spectra Physics) with a pulse duration of t ) 6 ns was
p
introduced through the fluorescence port into the rear aperture
Figure 2. Setup for fluorescent system for downstream confocal
detection of mixed fluid. The separation distance d between the site
of mixing and the site of fluorescent detection is ∼7 mm.
of a microscope objective (Zeiss Acroplan 40×, 0.8 NA or 20×,
pumped individually at a flow rate of 50 µL/h through a 200-µm-
wide channel and brought together at a Y junction. To monitor
the presence of fluorescent molecules within the channel, the
output of a continuous-wave argon ion laser at λ ) 488 nm (2214-
0
.5 NA). The focal volume of the objective was positioned within
the microfluidic channel at the interface between the two parallel
fluid streams. Laser pulse energies in the range of 20-25 µJ were
employed in all the experiments reported. The laser pulse
generated a plasma on the nanosecond time scale resulting in
the formation of a cavitation bubble that, upon collapse, produced
mixing of the adjacent fluid streams. Mixing dynamics were
visualized using a custom-built, time-resolved imaging setup that
1
0SL, JDS Uniphase) was coupled through a single-mode fiber
optic and reflected by a dichroic mirror into the back aperture of
a microscope objective (Nikon, 50×, 0.55 NA) that was screwed
into a custom-made filter block. The objective directed the argon
ion laser beam to the desired location within the microfluidic
channel. Any fluorescent emission collected by the microscope
objective was directed to a photomultiplier tube (PMT, R928,
Hamamatsu Photonics) that was also attached to the filter block.
A long-pass filter (LP 500, Edmund Scientific) was used to prevent
any argon ion laser light from reaching the PMT, and a 50-µm-
diameter pinhole was placed in front of the PMT to provide
confocal detection. This assembly was mounted on a three-axis
translation stage that was fixed to the microscope stage above
the microfluidic device. The translation stage allowed positioning
of the detection system at a defined distance downstream from
the microscope objective that delivered the pulsed Nd:YAG laser
radiation to produce mixing. Moreover, the translation stage
provided precise positioning of the focal volume of the argon ion
laser beam at the desired lateral and depth locations within the
channel. The fluorescence excitation volume provided by the
argon ion laser was positioned laterally on the “water side” of the
channel 20 µm from the channel wall and 80 µm from the water
fluorescein interface. This configuration ensured that fluorescence
would not be detected when no mixing was accomplished and
that appearance of a PMT signal indicated the presence of
fluorescein throughout nearly the entire width of the microfluidic
channel. The Nd:YAG laser was fired at a repetition rate of 0.4
Hz while the PMT monitored the fluorescence emission at 60 Hz.
Custom software routines written in Testpoint (Keithley Metra-
Byte) provided automated control over the delivery of Nd:YAG
laser pulses and PMT signal collection.
29,30
has been described previously
and employs the emission from
a fluorescent dye cell or an ultrashort duration flashlamp to
provide image illumination to the microscope condenser at the
desired delay time. A gated intensified CCD camera (PI-MAX,
Roper Scientific) was mounted on the microscope for image
capture. The camera gate duration was set to 0.5 ns when using
the fluorescent dye cell for illumination and 200 ns when using
the flashlamp illumination due to electronic jitter in the flashlamp
triggering. Thus, when using the flashlamp illumination, i.e., for
time delays longer than 3.6 µs, the exposure duration was
governed by the 40-ns duration of the flash lamp. WinView
software (Roper Scientific) was used to control relevant image
acquisition parameters such as gate delay and gate duration as
well as coordinating the delivery of the pulsed laser radiation and
the image illumination. Differential transmission of the illumination
pulse through the water and dye streams generated contrast and
1
7
allowed these dynamics to be imaged. Adjustments to image
brightness and contrast were performed subsequently using
Adobe Photoshop (Adobe Systems, Cupertino, CA) as necessary.
Each time point was imaged a minimum of three times and
representative images are shown in Results and Analysis.
Fluorescence System for Downstream Detection of Mixed
Dynamics. To examine the dynamics downstream from the site
of laser delivery, we utilized the same microfluidic configuration
described above but replaced the Naphthol Green dye solution
with a solution of 10 µM fluorescein and employed a fluorescence
detection system, as shown in Figure 2. The two fluids were
Fluorescence Detection of Amplex Red/Horseradish Per-
oxidase (HRP) Reaction. To demonstrate that the mixing
provided by this technique can be utilized to initiate an enzyme-
catalyzed biochemical reaction, we imaged the generation of
(
29) Rau, K. R.; Guerra III, A.; Vogel, A.; Venugopalan, V. Appl. Phys. Lett. 2004,
4, 2940-2942.
30) Rau, K. R.; Quinto-Su, P. A.; Hellman, A. N.; Venugopalan, V. Biophys. J.
006, 91, 317-329.
8
(
2
4486 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007

Figure 3. Time-resolved image series of cavitation bubble expansion and collapse and subsequent mixing effects produced by a single
nanosecond laser pulse in a 200-µm-wide microfluidic channel.
fluorescent resorufin resulting from mixing two nonfluorescent
reactant streams, one containing Amplex Red and the other
containing HRP and hydrogen peroxide. The two fluids were
pumped individually through 100-µm-wide channels and brought
together at a Y junction. Amplex UltraRed reagent, HRP, and 1
M Tris-HCL buffer were acquired from Invitrogen Corp. (Carlsbad,
recording using Power Director Pro 2.5ME software (CyberLink
USA, Fremont, CA).
RESULTS AND ANALYSIS
Time-Resolved Bright-Field Imaging of Laser-Induced
Mixing. Delivery of nanosecond Nd:YAG laser pulses focused
within the microfluidic channel induced plasma formation in the
fluid whose expansion resulted in shock wave emission and
cavitation bubble formation. The plasma formation and shock wave
2 2
CA). The 30% H O and dimethyl sulfoxide (DMSO) were acquired
from Sigma Chemical Co. (St. Louis, MO). All chemicals were
reagent-grade quality or better. A 10 mM stock solution of Amplex
Red was prepared in DMSO, and a 300 units/mL stock solution
of HRP was made using 0.1 M Tris-HCL buffer solution. Im-
mediately prior to use, the stock solutions were diluted in 0.1 M
Tris-HCL buffer solution to make one solution containing 250 µM
29,31,32
dynamics have been well-studied,
and here we focus on the
cavitation dynamics as it serves as the agent for the observed fluid
mixing. Figure 3 provides images from our time-resolved pho-
tography apparatus depicting the shock wave propagation, cavita-
tion bubble formation, expansion, and collapse at various time
points following the delivery of a single 20-µJ laser pulse within
the 200-µm channel. The time point is given in each frame, and
the flow is from viewer’s left to right. Figure 3a reveals a shock
wave resulting from the rapid plasma expansion and cavitation
bubble formation. The high pressure within the bubble causes
its initial rapid expansion that begins to slow measurably on a
Amplex Red and one solution containing 160 µM H
units/mL HRP. Each solution was metered using a 10-µL syringe
Gastight No. 1701, Hamilton Co.) connected to the device using
2 2
O and 1.6
(
Teflon tubing and driven by a syringe pump (Pump 11, Harvard
Apparatus) at a flow rate setting of 5 µL/h into a 100-µm channel
resulting in a mean flow velocity of 560 µm/s in the main channel.
Bright-field and fluorescence images of the enzyme reaction
were acquired using the Zeiss Axiovert microscope described
above equipped with a 100-W mercury lamp and band-pass filters
33
time scale of j100 ns. The bubble assumes an ellipsoidal shape
as its expansion is constrained by the channel walls (Figure 3c).
(λex ) 540 nm, λem ) 630 nm, Chroma Technology Corp.). The
laser was fired to generate mixing, and the production of resorufin
was imaged using a digital video camera (Panasonic GP-KR222)
and saved using a video cassette recorder (JVC HR-S7800U). Still
images at desired time points were captured from the taped
(31) Venugopalan, V.; Guerra III, A.; Nahen, K.; Vogel, A. Phys. Rev. Lett. 2002,
8
8, 078103.
(32) Vogel, A.; Nahen, K.; Theisen, D.; Noack, J. IEEE J. Sel. Top. Quantum
Electron. 1996, 2, 847-860.
(33) Vogel, A.; Busch, S.; Parlitz, U. J. Acoust. Soc. Am. 1996, 100, 148-165.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4487

Figure 4. Time-resolved image series for cavitation bubble dynamics and mixing within a 100-µm-wide microfluidic channel.
The ellipsoidal bubble reaches a maximum “length” of 240 µm
measured along its longest axis) at a time of 4.4 µs. The
maximum volume of the cavitation bubble is estimated as 1.04
nL, which corresponds to the volume of an ellipsoid with
dimensions of 240, 165, and 50 µm along its three axes.
After reaching its maximum size, the bubble collapses rapidly
with the reduction in the bubble size occurring faster in the
direction parallel with the channel walls. The rapid fluid inflow
associated with the bubble collapse results in bubble splitting
along the longitudinal axis of the microchannel (Figure 3d,e) and
produces counterpropagating jets toward the channel walls
patterns upon collapse within a microfluidic channel. In this case,
the mixing zone has a lateral extent of roughly 250 µm that
persists for ∼50 ms, after which restoration of the interface
between the two adjacent fluids begins and the region of mixed
fluid flows downstream and out of the field of view. Since we image
the dynamics in one plane, the total volume in which this fluid
jetting and mixing occurs is unknown. However, by approximating
the mixed region as an ellipsoid with dimensions equal to 250
µm × 200 µm × 50 µm, we can estimate the mixed volume of
fluid to be 1.3 nL.
Figure 4 shows the mixing dynamics within a 100-µm-wide
channel using a laser pulse energy of 25 µJ. The ellipsoidal bubble
shape is even more elongated due to the smaller channel cross
section and reaches a maximum length of 230 µm (Figure 4d).
The stronger confinement offered by the smaller channel reduces
the maximum bubble volume even more than that seen in Figure
3 even though a higher pulse energy was used to cause plasma
formation. Using measured dimensions of 230 µm × 110 µm ×
50 µm, we calculate a maximum ellipsoidal bubble volume of only
0.6 nL. Another interesting feature is the transient deformation
of the channel walls caused by the rapid bubble expansion as seen
in Figure 4c.
(
(Figures 3f-h). These jets produce a second bubble splitting event
and the formation of four very small bubbles that are convected
downstream. The appearance of the second bubble splitting event
suggests that these jets occupy the full height of the microfluidic
channel. These complex bubble dynamics disrupt the smooth
interface separating the two fluids and mix the adjacent fluid
streams. Upon bubble collapse, mixing is most evident in the
upper part of the channel due to a higher concentration of
Naphthol Green (Figure 3h,i). However, since the bubble collapse
is symmetric relative to the interface that initially separates the
two fluid streams, one can deduce that the mixing region is also
symmetric. At 300 µs, we observe the generation of symmetrical
whorls that are formed as a result of the impact of the fluid jet on
the channel walls. The spatiotemporal dynamics of the mixing
process are remarkably reproducible, demonstrating that laser-
generated cavitation bubbles produce consistent well-defined fluid
The features of the bubble collapse are slightly different from
those seen in Figure 3. The narrower channel enhances the speed
of the bubble collapse along the longitudinal axis of the channel
that again results in complete bubble splitting upon collapse
(Figure 4e,f). Again, we see counterpropagating jets toward the
4488 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007

Figure 6. Expanded view of one fluorescence signal peak from
Figure 5 with the corresponding Nd:YAG firing signal.
Figure 5. Fluorescent detection of mixing effects produced by Nd:
YAG laser pulses using the setup shown in Figure 2.
system and the 60-Hz sampling rate of the PMT signal, which
may be insufficient to adequately resolve the temporal fluctuations
in fluorescent intensity on the microscale.
bubble walls that occupy the full height of the microfluidic channel
and result in the second bubble splitting event. The jets produced
in the 100-µm-wide channel seem to produce more lateral mixing
upon bubble collapse (Figure 4h-k) than in the 200-µm-wide
channel. In this case, the mixed region extends laterally for a
distance of 200 µm and persists until the interface is restored at
Figure 6 provides an expanded plot of one fluorescence signal
peak from Figure 5 that follows the firing signal produced by the
Nd:YAG laser. The delay of 120 ms between the Nd:YAG firing
signal and the increase in the fluorescence signal is not meaningful
as it takes several seconds for the bolus of mixed fluid to travel
the several millimeters between the site of mixing and the site of
fluorescence detection. Thus, the fluorescence intensity peak that
is detected due to fluid mixing is not produced by the Nd:YAG
laser pulse whose firing signal is shown, but by a preceding laser
pulse. The width of the fluorescent peak (measured full width at
half-maximum) is 70 ms. This indicates that the bulk of the
detected fluorescein is concentrated in a bolus of mixed fluid ∼200
µm in length for the total volumetric flow rate of 100 µL/h in the
5
0 ms. Again we see that while the bubble collapse is complete
within 50 µs, the mixing dynamics persist for more than 15 ms.
Approximating the mixed fluid region by an ellipsoid with
dimensions 200 µm × 100 µm × 50 µm provides an estimate of
0
.52 nL for the mixed fluid volume.
Downstream Fluorescence Detection of Mixed Bolus of
Water and Fluorescein. The PMT-based fluorescence detection
system was employed to examine the characteristics of the mixed
bolus of fluid roughly 7 mm downstream from the region where
the Nd:YAG laser pulse was delivered. The focal volume of the
argon ion laser output used to excite fluorescence was placed at
a lateral position in the microfluidic channel where only water
was present so long as the parallel laminar flow of the water and
fluorescein solution remained undisturbed. Figure 5 shows the
result of one experimental run using this system. The first pair
of traces shown in Figure 5 provide signals for the firing of the
Nd:YAG laser used to produce the mixing and the measured signal
from the PMT when water was supplied to both inlets of the
microfluidic device. This shows that the presence of only water
in the channel results in no significant PMT signal even when
mixing is produced upstream by the firing of the Nd:YAG laser.
The second pair of traces show results when one of the water
streams is replaced by a 10 µM fluorescein solution. The firing of
each Nd:YAG laser pulse results in a spike in the PMT signal
indicating the presence of the fluorescein solution on the side of
the microfluidic channel where only water is usually present. This
result is indicative of the production of a mixed bolus of fluid that
occupies nearly the entire width of the channel and is convected
down the channel along with the main flow. Some pulse-to-pulse
variation in the peak intensity of the fluorescence signal measured
by the PMT is observed. This variation is likely due to the small
fluid volume interrogated by the confocal fluorescence detection
2
00 µm × 50 µm microfluidic channel. This dimension is roughly
consistent with the dimensions of the mixed bolus of fluid
visualized in our time-resolved imaging experiments. Close
examination of the PMT signal immediately surrounding the main
peak reveals a “ringing” pattern. This may indicate that a small
region is present between the main bolus of mixed fluid and
unmixed fluid where there are larger scale fluid structures of
fluorescein and water. In this experiment, a Nd:YAG laser pulse
repetition rate of 0.4 Hz was chosen in order to create a small
region of mixed fluid followed by an unmixed region. The duration
of the fluorescence peak suggests that continuous mixing within
the channel can be achieved by increasing the pulse repetition
rate of the Nd:YAG laser to 6-10 Hz.
Fluorescence Imaging of Amplex Red/HRP Reaction. To
demonstrate that the laser-induced mixing observed in the time-
resolved imaging studies is sufficient to initiate a biochemical
reaction within a continuous-flow microfluidic system, we used
this technique to initiate the HRP-catalyzed reaction between H
and Amplex Red. In the presence of HRP, Amplex Red reacts with
in a 1:1 stoichiometry, producing highly fluorescent resoru-
2 2
O
2 2
H O
34
fin. Figure 7a is a bright-field image at the intersection merging
(34) Zhou, M. J.; Diwu, Z. J.; Panchuk-Voloshina, N.; Haugland, R. P. Anal. Chem.
2004, 76, 3694-3698.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4489

Figure 7. Image series demonstrating the production of fluorescent resorufin using the proposed mixing technique to initiate the HRP-catalyzed
reaction between Amplex Red and hydrogen peroxide.
Figure 8. Quantitative analysis of the fluorescent images in Figure 7 demonstrating that the fluorescent product resorufin is produced throughout
the entire width of the microfluidic channel and is convected downstream with the main hydrodynamic flow.
the two nonfluorescent streams: H
and Amplex Red in the bottom stream. Figure 7b provides a
background” fluorescence image prior to delivery of the laser
2
O
2
and HRP in the top stream
between the two vertical solid lines shown in Figure 7b, d, and e.
The fluorescent intensities shown are those determined from
Figure 7d and e normalized against the “background” intensity
of Figure 7b. These traces clearly show that the fluorescent
product of the reaction occupies the full width of the channel.
Figure 8b is an integrated measure of the fluorescent intensity as
a function of horizontal position along the length of the channel
between the two horizontal dashed lines shown in Figure 7b and
d-f. This figure illustrates the minimal fluorescence intensity
before and 2 s following the laser delivery as well as the
propagation of the reaction product downstream on the subsecond
time scale.
“
pulse. Figure 7c provides the fluorescence image at the time of
laser pulse delivery (t ) 0) showing the fluorescence produced
by the plasma emission. Panels d and e in Figure 7 are
fluorescence images taken t ) 0.33 and 0.5 s following the laser
pulse delivery, respectively. One can easily identify a “bolus” of
fluorescence that corresponds to the formation of the reaction
product that occupies the entire width of the channel and is
convected downstream by the main flow. Figure 7f is the
fluorescence image at t ) 2 s following the laser pulse delivery
and bears a strong similarity to Figure 7b since the bolus of the
fluorescent reaction product has been convected downstream
and out of the field of view. This time scale for the flow of the
reaction product out of the field of view and the re-establishment
of parallel laminar flow is consistent with the mean flow velocity
of 560 µm/s.
DISCUSSION
These time-resolved imaging and fluorescence methods have
enabled the examination of both the hydrodynamic events that
lead to the formation of a small volume of mixed fluid and its
downstream convection in the microfluidic channel on nanosecond
to second time scales. The initiating event for the mixing process
is the formation of a laser-induced plasma within the aqueous
medium. Plasma formation is a nonlinear process and does not
rely on linear optical absorption by the aqueous medium. Thus,
the proposed method is applicable at any optically accessible
To provide a more quantitative assessment of the spatial
distribution of the fluorescent reaction product within the channel,
we provide the spatial distribution of the fluorescent intensities
in Figure 8. Figure 8a is an integrated measure of the fluorescent
intensity as a function of vertical position within the channel
4490 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007

location within a microfluid device. The process of laser-induced
plasma formation in water is a well-studied phenomenon whose
lifetime, for nanosecond pulse widths, is governed by the duration
of the laser pulse.32 The rapid plasma expansion results in
cavitation bubble formation, expansion, and collapse; processes
that are all clearly visualized within the microfluidic channel. The
confinement offered by the microfluidic channel results in
maximum bubble volumes that are smaller than those observed
for expansion in a free medium. The confinement also results in
bubble splitting and jet formation upon bubble collapse that
accomplishes the fluid mixing. While cavitation bubble dynamics
a high numerical aperture objective at the desired location. The
bubble collapse was found to produce flow patterns that were
reproducible on both microsecond and millisecond time scales.
Since flow patterns produced during ultrasound mixing in micro-
channels have not been studied, it is not known whether similar
processes are operative in these methods.
The production of cavitation bubble-induced mixing through
laser-induced plasma formation may be cause for concern due to
the potential for direct damage by the plasma. While the plasma
itself can reach high temperatures,42 the plasma is typically very
small (∼10-µm diameter), has a short lifetime (∼20 ns), and cools
rapidly upon expansion. Nonetheless, it is likely that an amount
of fluid equivalent to the plasma volume is thermally inactivated.
Experimental studies show that, for the laser parameters used in
this study, the plasma volume is well-described by an ellipsoid
∼13 µm in length and ∼8 µm in diameter resulting in a plasma
3
5,36
in the proximity of single rigid or elastic boundaries
as well
as bubble-bubble interactions within microfluidic channels37 have
been studied, we believe this is the first examination of the
dynamics of a single cavitation bubble within a channel whose
dimensions are similar in size to the bubble itself. The hydrody-
namic resistance offered by the microfluidic channel walls results
in the more rapid collapse of the bubble surfaces that are not
adjacent to the channel walls. This leads to asymmetric bubble
collapse, specifically invagination or “pinching” of the bubble wall
in the midplane of the channel, followed by jet formation and a
second bubble splitting event. While the impact of cavitation-
induced jets have been known to cause pitting damage in metal
3
1,43
volume of ∼0.7 pl.
This suggests that there is over a 1000:1
ratio between the volume of mixed fluid (∼1 nL) to the volume
of reactants destroyed by plasma formation. Moreover, even if
the entire energy of the 20-µJ laser pulse is confined to the mixed
fluid volume on the order of 1 nL, the resulting temperature rise
is no more than 5 deg kelvin. Our successful demonstration of
2 2
the HRP-catalyzed reaction of Amplex Red and H O resulting in
3
8
films and tissue puncture in ophthalmic surgery, we do not
observe mechanical damage due to the low laser pulse energies
used and the mechanical resiliency of PDMS. Instead, upon hitting
the channel walls, the jet flows outward, leading either to whorl
formation in the case of the 200-µm channel or more complex
patterns seen in the case of the 100-µm channel. The bubble
collapse dynamics also caused stretching and folding of fluid
elements, characteristics necessary for good micromixing. We also
observed that the mixing region extended to regions upstream
of the flow leading to “active mixing”, defined as a process in
which the fluid interfaces interact with the flow and modify it.39
Clearly, the imaging system employed here could find general
use for the examination and analysis of other fast biophysical
effects in microfluidic devices.40
resorufin demonstrates that any generated heat did not result in
significant inactivation of the reactants, catalyst, or product. One
potential limitation of this technique is that the plasma does indeed
vaporize a small volume of fluid thereby leading to the generation
of vapor bubbles that persist after the cavitation bubble collapse.
Although we did not encounter problems when operating the Nd:
YAG laser at a pulse repetition rate of 0.4 Hz, higher pulse
repetition rates may lead to the generation of large numbers of
vapor bubbles that could block the channel. This can be remedied
by the use of shorter wavelengths (e.g., λ ) 355 nm) or shorter
44,45
laser pulse durations (ps or fs).
Reductions in wavelength or
pulse duration enable the formation of a laser-induced plasma at
lower pulse energies (e1 µJ/pulse) with less energy available for
vaporization. However, this may also lead to a smaller amount of
bubble energy, leading to less violent bubble collapse and the
production of smaller volumes of mixed fluid. Another approach
could be surface modification of the channel walls to make them
more hydrophilic and less prone to bubble sticking. Clearly the
promising results of this study need to be followed up with a
detailed examination of the dependence of the fluid mixing process
on laser pulse duration, pulse energy, wavelength, and pulse
repetition rate.
Although mixers based on the use of ultrasound to generate
2
1,22,41
cavitation bubbles have been described,
there are several
important differences between our method and those that employ
ultrasound. In ultrasonic mixers, an acoustic wave is launched
into the medium by a piezoelectric transducer that is integrated
onto the device. Mixing occurs due to microflows and eddies set
up by cavitation microstreaming. In our method, the localized flow
instability and mixing is produced by a single bubble, as opposed
to the formation of multiple bubbles as is the case for cavitation
microstreaming. The site of bubble formation (and hence mixing)
can be accurately controlled by focusing the laser microbeam with
CONCLUSION
We have demonstrated a novel technique for mixing two
streams within a microfluidic channel using laser-generated
cavitation bubbles. Time-resolved imaging allowed visualization
of the complex fluid patterns produced upon bubble collapse.
While the cavitation bubble formation expansion and collapse was
(
(
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37) Chen, Y. H.; Chu, H. Y.; I, L. Phys. Rev. Lett. 2006, 96, 034505.
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Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4491

complete on a time scale of ∼25 µs, the restoration of laminar
flow did not occur until 50 ms following the laser pulse delivery.
The cavitation bubble dynamics disrupted the parallel laminar flow
and led to the formation of a local volume of mixed fluid. We
estimated this mixed volume to be in the range of 0.5-1.5 nL.
Fluorescence detection downstream of the mixing site confirmed
that the delivery of each Nd:YAG laser pulse resulted in the
formation of a mixed bolus of fluid that occupied the entire channel
and was convected downstream with the main flow. We also used
fluorescence video microscopy to demonstrate that laser-induced
mixing can be used to initiate an enzyme-catalyzed reaction within
a microfluidic system. This method of mixing using laser-
generated cavitation bubbles may be particularly attractive for
microfluidic applications since no modifications need be made to
the microfluidic channel geometry and there is complete flexibility
in the location of the mixing site. Moreover, the time-resolved
imaging system detailed in this report could be used for the
visualization of other fast phenomena in microfluidics.
ACKNOWLEDGMENT
We thank Prof. Mark Bachman for helpful discussions. We
are grateful to the NIH for support under Grant R01-EB04436 and
the Laser Microbeam and Medical Program (#P41-RR01192).
Support is also provided by the University of California System-
wide Biotechnology Research and Education Program (UC BREP)
GREAT Training Grant #2006-12.
Received for review January 12, 2007. Accepted April 6,
2007.
AC070081I
4492 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007