111111111111111111111111111111111111111111111111111111111111111111111111111 

US006907895B2 

(12) United States Patent (10) Patent No.: US 6,907,895 B2 

Johnson et ai. (45) Date of Patent: Jun. 21,2005 

(54) METHOD FOR MICROFLUIDIC FLOW 

MANIPULATION 

(75) Inventors: Timothy J. Johnson, Charlestown, MA 

(US); David J. Ross, Silver Spring, 

MD (US); Laurie E. Locascio, North 

Potomac, MD (US) 

(73) Assignee: The United States of America as 

represented by the Secretary of 

Commerce, Washington, DC (US) 

( *) Notice: Subject to any disclaimer, the term of this 

patent is extended or adjusted under 35 

U.S.c. 154(b) by 138 days. 

(21) Appl. No.: 10/188,664 

(22) Filed: Jul. 1,2002 

(65) Prior Publication Data 

US 2003/0051760 A1 Mar. 20, 2003 

(60) 

Related U.S. Application Data 

Provisional application No. 60/323,509, filed on Sep. 19, 

200l. 

(51) 

(52) 

(58) 

Int. CI? ................................................. BOiF 13/02 

U.S. CI. ........................... 137/1; 137/896; 366/341; 

366/348 

Field of Search ............................ 137/1, 896, 897, 

137/898; 366/341, 348 

(56) References Cited 

U.S. PATENT DOCUMENTS 

3,226,036 A * 12/1965 Grahn ........................ 366/101 


3,788,557 A * 1/1974 Breunsbach ................ 137/897 


5,900,130 A * 5/1999 Benvegnu et al. ..... 366/DIG. 2 

OTHER PUBLICATIONS 

Armand Ajdari, Transverse electrokinetic and microfluidic 


effects in micro patterned channels: Lubrication analysis for 


slah geometries, The American Physical Society, Physical 


Review E, vol. 65,016301. 


Abraham D. Stroock, Stephan K. W. Dertinger, Armand 


Ajdari, Igor Mezic, Howard A. Stone, George M. White­


sides, Chaotic Mixer for Microchannels Science, vol. 295, 


Jan. 25, 2002, pp. 647-651. 


* cited by examiner 

Primary Examiner-Stephen M. Hepperle 

(74) Attorney, Agent, or Firm-Cochran Freund & Young 

LLC 

(57) ABSTRACT 

Disclosed is an apparatus and method for the mixing of two 

microfluidic channels wherein several wells are oriented 

diagonally across the width of a mixing channel. The device 

effectively mixes the confluent streams with electrokinetic 

flow, and to a lesser degree, with pressure driven flow. The 

device and method may be further adapted to split a pair of 

confluent streams into two or more streams of equal or 

non-equal concentrations of reactants. Further, under electrokinetic flow, the surfaces of said wells may be specially 

coated so that the differing electro osmotic mobility between 

the surfaces of the wells and the surfaces of the channel may 

increase the mixing efficiency. The device and method are 

applicable to the steady state mixing as well as the dynamic 

application of mixing a plug of reagent with a confluent 

stream. 

7 Claims, 20 Drawing Sheets 

INLET 

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INLET 

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FIGURE 10 

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u.s. Patent JUll.21,200S Sheet 14 of 20 US 6,907,895 B2 

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u.s. Patent Jun. 21,2005 Sheet 15 of 20 US 6,907,895 B2 

CROSS SECTION. CROSS SECTION CROSS SECTION 

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CROSS SECTION CROSS SECTION CROSS SECTION 


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FIGURE 15 

u.s. Patent JUll.21,200S Sheet 18 of 20 US 6,907,895 B2 

FIGURE 16A 


0.11 " '. 

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CONCENTRATION (mM) 

0.05 

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CCD 

DETECTOR 

MIRROR 6 

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5 BEAM 

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US 6,907,895 B2 


1 


METHOD FOR MICROFLUIDIC FLOW 


MANIPULATION 


CROSS REFERENCE TO RELATED 

APPLICATIONS 

This application is based upon and claims priority to U.S. 

provisional application 60/323,509 entitled "MICROCHANNEL DESIGNS FOR MIXING AND SPLITTING 

MICROFLUIDIC STREAMS UNDER ELECTROKINETIC OR PRESSURE DRIVEN FLOW" filed Sep. 19, 

2001 by Timothy J Johnson, David J Ross, and Laurie E 

Locascio, the entire disclosure is specifically incorporated 

herein by reference for all that it discloses and teaches. 

BACKGROUND OF THE INVENTION 

a. Field of the Invention 15 

The present invention pertains generally to micro fluidic 

flow devices and specifically to mixers and splitters of 

microfluidic flow. 

b. Description of the Background 

The application of micro fluidic analytical devices to 

chemical or biological assays has developed rapidly over the 

last decade. Although microfluidic devices have been highly 

successful, several performance limitations exist, notably 

reagent mixing. 

Most mixing devices rely on diffusive mixing, wherein 25 

the natural laminar flow effects and the reagent's inherent 

diffusion coefficient cause the reagents to mix. Therefore, 

the mixing chamber/channel is usually extended to lengths 

that will ensure a completely mixed outlet stream. This 

approach may be acceptable for low flowrates, but high 

flowrates (>1 cm/s) or low analyte diffusion coefficients 

«10-7 cm/s2) will require excessively long mixing channels. The difficulty in rapidly mixing reagents results from 

the fact that the system is restricted to the laminar flow 

regime (Re<2000) and also because the feature sizes are too 35 

small (typically <100,um) to incorporate conventional mixing mechanisms. 

The lack of turbulence in micro fluidic systems has led to 

device designs that utilize multi-laminate, or flow splitting 

techniques to accomplish mixing in channels of shorter 

length. These designs split the incoming streams into several 

narrower confluent streams to reduce the mixing equilibrium 

time. Once mixing is complete, the narrow channels are then 

brought back together into a larger main channel for further 

transport, processing, and/or detection. The effectiveness of 45 

the flow splitting concept is based on the fact that the 

equilibrium time scales quadratically with the width of the 

channel. For example, if the width of the channel decreases 

by two, then the equilibrium time and the channel length 

decreased by a factor of four, or 25% of the original length. 

However, even a mixing length of 25% may still be unsuitable for some applications. 

Other techniques for mixing may rely on active mechanical mixing, such as stirring paddles and the like. For very 

small fluidic passages, such devices are extremely fragile 55 

and difficult to manufacture. 

It would therefore be advantageous to provide a device 

and method of mixing two confluent micro fluidic laminar 

flows that did not require an excessively long channel to 

effectively mix the flows. Further, it would be advantageous 

to provide a splitting mechanism that may be able to split a 

stream of reagents into two streams of differing concentrations. 

SUMMARY OF THE INVENTION 65 

The present invention overcomes the disadvantages and 

limitations of the prior art by providing a device and method 

2 

for effectively mixing two confluent laminar reagents within 

a very short stream length. This is accomplished by passing 

the confluent laminar flows over a series of narrow wells that 

are angled across the width of the channel. The device and 

method may also be used to split incoming streams into 

multiple streams of equal or non-equal proportions. 

Additionally, the present invention may be used for the 

mixing of plugs of reagents while minimizing axial dispersion of the reagent plug. 

The present invention may therefore comprise a mixer of 

laminar microfluidic streams propelled by electrokinetic 

flow comprising: a first inlet channel; a second inlet channel; 

a mixing channel starting at the confluence of the first inlet 

channel and the second inlet channel; and a plurality of wells 

disposed in the mixing channel, the wells being obliquely 

oriented substantially across the width of the mixing channel. 

The present invention may further comprise a splitter of 

a substantially laminar microfluidic stream comprising: a 

splitting channel coupled to at least two inlet ports and at 

least one outlet port in which the substantially laminar 

microfluidic stream has an axis of flow; and a plurality of 

wells disposed in the splitting channel, the wells being 

oriented substantially longitudinally across the width of the 

channel and diagonally across the axis of flow, the wells 

being deeper in profile than in width. 

The present invention may further comprise a method of 

mixing two confluent laminar flows in microchannels comprising: providing a first inlet stream and a second inlet 

stream that meet at a confluence point to produce a confluent 

stream; passing the confluent stream through a mixing 

channel, the mixing channel comprising a plurality of wells, 

the wells being oriented substantially longitudinally across 

the width of the mixing channel and diagonally across the 

mixing channel, the wells being deeper in profile than in 

width; and producing a mixed laminar flow at the output of 

the mixing channel. 

The advantages of the present invention are that flows 

may be combined and mixed without the conventional long 

lengths of diffusive mixing. The device may be further 

adapted to create two or more streams of equal or non-equal 

proportions of reagents. The device may be adjusted to tune 

the mix of the flows by adding various wells at different 

orientations, depths, and with various electroosmotic mobility coatings, all of which may have a substantial effect on the 

performance of the present invention. 

BRIEF DESCRIPTION OF THE DRAWINGS 

In the drawings, 

FIG. 1 is an illustration of an embodiment of the present 

invention of a micro fluidic mixer. 

FIG. 2Ais a white light microscopy image of the embodiment of FIG. 1. 

FIG. 2B is an image of the fluorescence of Rhodamine B 

introduced in the first inlet mixed with the buffer solution 

introduced in the second inlet. 

FIG. 2C is a similar image as FIG. 2B, except the flow is 

0.81 cm/s. 

FIG. 3 illustrates experimental results of the degree of 

mixing of the embodiment of FIG. 2A, wherein an electroosmotic flow of 0.06 cm/s was achieved. 

FIG. 4 illustrates the same experimental set up as FIG. 2C, 

with the electroosmotic flowrate of 0.81 cm/s. 

FIG. 5 illustrates the same experimental set up of FIGS. 

3 and 4, with a pressure driven flow. 
10 

20 

30 

40 

50 

60 

US 6,907,895 B2 


3 

FIG. 6 illustrates a second preferred embodiment of the 

present invention. 

FIG. 7 is a white light microscopy image of the embodiment of FIG. 6. 

FIG. 8 illustrates experimental results of the degree of 

mixing of two reagents using embodiment of FIG. 7 and an 

embodiment similar to embodiment of FIG. 7 but with three 

wells instead of four. 

FIG. 9 illustrates the results of the same experimental set 

up as FIG. 8 with a higher electro osmotic flowrate of 0.81 

cm/s. 

FIG. 10 illustrates an embodiment of a stream splitter 

wherein two inlet ports form a confluent stream wherein the 

fluid on one half of the channel is split into two streams 15 

located on opposite sides of the channel that then exit 

through two outlets. 

FIG. 11 illustrates an embodiment of a four-well mixer 

that was analyzed with computational fluid dynamics techniques for variations in the present invention. 

FIG. 12 illustrates some computational analysis of the 

flow patterns for various depths of wells, based on the 

embodiment shown in FIG. 11. 

FIG. 13 illustrates some computational results of various 

angles of the walls, based on the embodiment of FIG. 11. 25 

FIG. 14A illustrates a plan view of the flow pattern of an 

embodiment of the present invention of a mixer with quantity 4 wells oriented at 15 degrees off of the axis of flow. 

FIG. 14B illustrates a cross sectional view of the flow 

pattern of FIG. 14A, as observed from the cross section 

E-E. 

FIG. 15 illustrates some computational results of changes 

in the electro osmotic (EO) mobility of the surfaces of the 

wells. 35

FIG. 16A illustrates a plug of fluid introduced to and 

transported by a channel. 

FIG. 16B illustrates an embodiment of the present invention wherein a plug of fluid is introduced into a channel in 

which four wells are disposed. 

FIG. 17 illustrates some results of a computational analysis of the flow of the embodiments of FIGS. 16A and 16B. 

FIG. 18 illustrates the laser apparatus used to manufacture 

the experimental devices referenced in the specification. 45 

DETAILED DESCRIPTION OF IRE 

INVENTION 

FIG. 1 illustrates an embodiment 100 of the present 

invention of a micro fluidic mixer. Two inlet streams 102 and 

104 are combined and mixed in the mixing region 106 to 

produce a mixed flow that exhausts out of the outlet 108. The 

mixing region 106 comprises several wells 110, 112, 114, 

116, 118, 120, 122, 124, 126, and 128 that are recessed into 

the outlet 108. 55 

In a first embodiment, the channels have a uniform, 

trapezoidal cross section, with the width at the top being 72 

,urn, depth 31 ,urn, and the width at the bottom being 28,um. 

The wells 110, 112, 114, 116, 118, 120, 122, 124, 126, and 

128 have a depth of 85 ,urn in the center of the well. 

The wells 110, 112, 114, and 116 are parallel to each other 

and uniform in size. The wells are angled approximately 45 

degrees from the axis of the outlet 108. The wells 118, 120, 

122, 124, 126, and 128 are perpendicular to each other and 

are approximately 45 degrees from the axis of the outlet 108. 65 

The flow of reagents through the embodiment 100 may be 

electrokinetic, electroosmotic, or pressure driven flow. Since 

4 

the electroosmotic flow is a wall driven phenomenon based 

on the surface charge of the microchannel wall and on the 

local electric field, the fluid may enter and follow the 

contour of the wells, with the slanted well design used to 

induce lateral transport across the channel. In a pressure 

driven flow, the flow is not a wall driven phenomenon and 

therefore the fluid is not forced to enter the wells like 

electroosmotic flow. However, the presence of the wells 

induces some lateral transport across the channel in pressure 

driven flow, although not as effective as with electro osmotic 

flow. The presence of the wells under electrokinetic flow, 

being the combination of both electroosmotic flow and 

electrophoretic flow, will also enhance the mixing. 

In an experiment with the present embodiment, a confluence of Rhodamine B in a carbonate buffer was introduced 

into inlet 102 and carbonate buffer was introduced into inlet 

104. The fluorescence of the Rhodamine B was measured to 

indicate the degree of mixing achieved by the embodiment 

100. 

The method of manufacture of the embodiment used in 

the experiment, as well as the experimental setup and 

method of measurements, are given elsewhere in this specification. 

For the experiments, the length of each channel arm was 

0.8 cm long, and the dimensions of the channel are 72 ,urn 

wide at the top, 28 ,urn wide at the bottom, and 31 ,urn deep. 

The laser etched wells that spanned across the entire width 

of the channel had a depth of 85 ,urn in the center of the well 

relative to the bottom of the imprinted channel. The intensity 

measurement was taken at distance 130 or 443 ,urn from the 

beginning of the confluent region. 

FIGS. 2A-C illustrate the results of the experiments as 

detailed elsewhere in this specification. FIG. 2A is a white 

light microscopy image of the embodiment of FIG. 1. FIG. 

2B is an image of the fluorescence of Rhodamine B introduced in the first inlet 202 mixed with the buffer solution 

introduced in the second inlet 204, producing a mixed 

stream 206. The flowrate is 0.06 cm/s. FIG. 2C is a similar 

image as FIG. 2B, except the flow is 0.81 cm/s. 

FIG. 3 illustrates the degree of mixing of the embodiment 

100, wherein an electroosmotic flow of 0.06 cm/s was 

achieved. The horizontal axis is the position across the width 

of the outlet 108 and the vertical axis is the normalized 

intensity of the fluorescence of the Rhodamine B. The curve 

302 represents the results of the measurement taken with no 

mixing wells present. The curve 304 represents perfect 

mixing. Curve 304 is trapezoidal in shape, following the 

profile of the trapezoidal outlet 108. The curve 306 represents the actual experimental results. The details concerning 

the experimental procedure and equipment used to perform 

all of the experiments referenced in this specification are 

given elsewhere in this specification. 

FIG. 4 is a graph that illustrates the same experimental set 

up as FIG. 3, with the electroosmotic flowrate of 0.81 cm/s. 

The line 402 represents the results of the measurement taken 

with no mixing wells present. The line 404 represents 

perfect mixing. The line 406 represents the actual experimental results. 

From the results illustrated in FIGS. 2B, 2C, 3, and 4, the 

degree of mixing exiting the mixer was 87.2% and 80.5% 

respectively, for the flowrates of 0.06 cm/s and 0.81 cm/s. To 

achieve the same degree of mixing, theoretical predictions 

state that a channel length of 0.2 cm and 2.3 cm for 

electroosmotic flowrates would be required if no mixer were 

present and based on diffusional mixing, assuming that the 

diffusion coefficient of the fluorescent material, Rhodamine 
10 

20 

30 

40 

50 

60 

US 6,907,895 B2 


5 6 


B, is 2.8xlO- 6 cm2/s. These results indicate that the length 

of the present embodiment is 22% and 2% of the length of 

a comparable diffusive mixer for the present application. 

FIG. 5 is a graph that illustrates the results of the same 

experimental set up of FIGS. 2, 3, and 4, with a pressure 

driven flow. The measurements were taken at 443 ,urn from 

the beginning of the confluence region. The curve 502 

represents the perfect mixing results. The curve 504 represents the experimental results with a pressure driven flow at 

0.21 cm/s. The curve 506 represents the experimental results 

with a pressure driven flow at 1.25 cm/s. Since the pressure 

driven flow is not a wall driven phenomenon and therefore 

the fluid is not forced to enter the wells like electroosmotic 

flow, the effects of the mixing are not as great with pressure 

driven flow as with electro osmotic flow. However, the 15 

presence of the wells does introduce some lateral transport 

across the channel. This suggests that a series of wells could 

be optimized for mixing under pressure driven flow. 

FIG. 6 illustrates a second embodiment 600 of the present 

invention. Two inlet streams 602 and 604 are combined and 

mixed in the mixing region 606 to produce a mixed flow that 

exhausts out of the outlet 608. The mixing region 606 

comprises several wells 610, 612, 614, and 616 that are 

recessed into outlet 608. The measurement region 618 is 183 

,urn from the point of confluence of the inlets 602 and 604. 25 

The shape and dimensions of the channels and wells are the 

same as with embodiment 100 of FIG. 1. The wells 610, 612, 

614, and 616 are parallel to each other. 

FIG. 7 is a white light microscopy image of an example 

of embodiment 600. 

FIG. 8 is a graph that illustrates the degree of mixing of 

two reagents using embodiment 600 and an embodiment 

similar to embodiment 600 but with three wells instead of 

four. The experimental results of FIG. 8 show results for 35 

electroosmotic flow of 0.06 cm/s taken 183 ,urn from the 

point of confluence of the inlet flows. The horizontal axis is 

the position across the width of the outlet stream 608 and the 

vertical axis is the normalized intensity of Rhodamine B. 

The same experimental setup was used for the results of 

FIG. 3 as FIG. 8, with the differences being the configuration 

of the mixing region and the position of the measurements. 

The curve 802 represents the mixing profile of the two 

inlet streams when no mixing wells are present. The curve 

804 represents the perfect mixing of the two streams. The 45 

curve 806 represents the mixing profile for the electro osmotic flowrate of 0.06 cm/s and a three well mixer. The 

curve 808 represents the mixing profile for the same flowrate 

and a four well mixer. The three well mixer is the embodiment 600 with well 616 removed. 

FIG. 9 illustrates the results of the same experimental set 

up as FIG. 8 with a higher electro osmotic flowrate of 0.81 

cm/s. The curve 902 represents the mixing profile of the two 

inlet streams when no mixing wells are present. The curve 

904 represents the perfect mixing of the two streams. The 55 

curve 906 represents the mixing profile for a three well 

mixer. The curve 908 represents the mixing profile for a four 

well mixer. The three well mixer is the embodiment 600 with 

well 616 removed. 

The curve 908 forms two distinct humps, 910 and 912, 

indicating that the four well mixer may be able to split the 

incoming streams into two streams of similar concentrations. The number of wells, the shape, dimension, and 

placement of the wells may be adapted to provide different 

dilutions of the incoming fluid. Such adaptations may 65 

depend on the reagents and the diffusivity constants of the 

various components of the confluent streams. As such, the 

particular result desired, such as splitting a stream or mixing 

a pair of confluent streams may be obtained by adjusting the 

quantity and position of the various wells. 

FIG. 10 illustrates an embodiment 1000 of a stream 

splitter wherein inlet port 1002 and inlet port 1003 form a 

confluent stream wherein the fluid that is on one half of the 

channel is split into two streams due to the presence of the 

slanted wells, where the split streams are located on opposite 

sides of the channel that then exit through outlet 1004 and 

outlet 1006. The flow of the micro fluidic stream passes over 

three wells 1008, 1010, and 1012 of similar design and 

construction as those of other embodiments described in the 

present specification. 

In lab-on-a-chip or ,u-TAS (micro Total Analysis 

Systems), the use of a series of wells within a microchannel 

may greatly enhance the effectiveness of the entire system, 

especially when the system is limited to the laminar flow 

regime. The present invention is effective for low flowrates 

(<1 cm/s) as well as high flowrates (>1 cm/s). The present 

invention is further able to effectively mix flows that are 

driven electrokinetically, electroosmotically, or by pressure. 

Several embodiments of the present invention may be 

used in series, such that one stream is separated and split, 

then separated and split again, with the end result being 

several outlet streams with differing dilutions of the original 

incoming stream. Such a system is known as serial dilution. 

FIG. 11 illustrates an embodiment 1100 of a four-well 

mixer that was analyzed with computational fluid dynamics 

techniques for variations in the present invention. The 

computational analyses were designed to correspond to the 

experimental results shown in the previous figures. The 

channel geometry and fluid properties were selected to 

closely match those of the experiments. The inlet 1102 

contains a buffer fluid with Rhodamine B that is mixed with 

a second inlet 1104 that contains only the buffer fluid. The 

fluid exits the embodiment 1100 via the outlet 1105. The four 

wells 1106, 1108, 1110, and 1112 are located at an angle 

theta 1113 from the centerline axis of the mixer. The 

geometry of the embodiment 1100 is similar to the previous 

embodiments described herein. Cross-section line A 1114 

will be used to illustrate the incoming streams prior to 

mixing. Cross-section line B 1116 will be used to illustrate 

the mixing of the streams while in the well 1112, the last of 

the four wells. Cross-section line C 1118 will be used to 

illustrate the mixing of the streams 5 ,urn past the exit of the 

last well. Cross-section line D 1120 will be used to illustrate 

the mixing of the streams at a location of 420 ,urn past the 

point of confluence. 

FIG. 12 illustrates some computational analyses of the 

flow patterns for various depths of wells, based on the 

embodiment 1100 shown in FIG. 11, with a constant well 

angle of 45°. The results for cross section A 1202 illustrate 

the two incoming flows 1204 and 1206 prior to mixing. The 

results for cross section B 1208 illustrate the flow patterns 

for the flow within the last of the four wells. The 10 ,urn 

depth results 1210 show that very little of the mixing is 

occurring in the well. The 50 ,urn depth results 1212 show 

that a substantial portion of the mixing is occurring in the 

well. The 85 ,urn depth results 1214 show that a substantial 

portion of the mixing is occurring in the well, but that there 

is not much increase in the mixing due to the larger depth 

over the 50,um results 1212. These results indicate that there 

is a finite depth wherein increasing the depth does not 

increase the degree of mixing substantially. Further, these 

results illustrate that the wells greatly affect the mixing by 

forcing the fluids to fold over each other. 
10 

20 

30 

40 

50 

60 

US 6,907,895 B2 


7 

FIG. 13 illustrates the results of various angles of the 

wells as represented by the angle theta 1113 of FIG. 11. For 

all well angles illustrated, the depth of the wells was held 

constant at 50 ,urn below the bottom of the imprinted 

channel. The results of cross section B-B 1302, cross 

section C-C 1304, and cross section D-D 1306 are shown 

in columns. The results for the various angled wells are 

shown in rows. Results along row 1308 are for wells at a 

right angle or 90 degrees to the axis of flow. Results along 

rows 1310, 1312, and 1314 are for wells at 60 degrees, 30 

degrees, and 15 degrees to the axis of flow. The results 

indicate that a decreased angle of the well achieves a higher 

degree of mixing. 

The results along row 1308 for right angle wells show that 

there is no lateral transport across the width of the well. As 15 

the angle of the wells is decreased, there is increased lateral 

transport to the point where the flow may be folded over on 

top of itself more than once. The folding action is an 

important mechanism that causes efficient mixing. 

FIG. 14A illustrates a plan view of the flow pattern of an 

embodiment 1400 of a mixer with quantity 4 wells oriented 

at 15 degrees off of the axis of flow, and well depths set to 

50,um below the bottom of the imprinted channel. FIG. 14B 

illustrates a cross sectional view of the flow pattern of FIG. 

14A, as observed from the cross section E-E. The flow 25 

lines 1402 and 1404 illustrate that the fluid may exit the first 

well 1406 and reenter another well 1408 and thereby may 

fold during the passage through the mixer 1400. 

FIG. 15 illustrates the results of changes in the electro osmotic (EO) mobility of the surfaces of the wells. Different 

manufacturing processes may create different EO mobilities 

on various surfaces of the channels. For example, of the 

manufacturing processes described for the experiments 

described elsewhere in this specification, imprinting a chan- 35 

nel has been shown to yield a different EO mobility than the 

laser ablation manufacturing method. Further, other methods 

such as polyelectrolyte multilayers, surface chemistry 

modifications, EO mobility suppression coatings, and other 

methods may be used individually or in combination to 

selectively change the EO mobility of selective surfaces of 

the mixer. 

The results of FIG. 15 illustrate the effects of increasing 

the EO mobility of the surfaces of the wells with respect to 

the EO mobility of the remaining surfaces of the mixer. The 45 

results are for a four well mixer with 45 degree wells at a 

depth of 50 ,urn below the bottom of the imprinted channel. 

The column 1502 illustrates the results for section B-B, 

column 1504 illustrates the results for section C-C, and 

column 1506 illustrates the results for section D-D, all of 

which relate the cross sections illustrated in FIG. 11. 

For the purposes of this discussion, a ratio of the EO 

mobility of the wells divided by the EO mobility of the 

remainder of the surfaces will be rEOM' The row 1508 

illustrates the results when rEOM is 1.24. Row 1510 illus- 55 

trates the results for rEOM of 2.00 and row 1512 illustrates 

the results for rEOM of 3.00. Row 1508 is illustrative of the 

approximate rEOM of the experimental results described in 

FIGS. 7, 8, and 9. The results indicate that as the rEOM is 

increased, mixing can be enhanced. In other words, the 

increase of the EO mobility, by different manufacturing 

processes, selectively applied coatings, or other methods 

may dramatically increase the performance of a mixer of the 

present invention. 

A use for the present invention is the mixing of plugs of 65 

fluid. Applications for such a use may be for lab on chip 

applications wherein several samples of fluid may be ana8 

lyzed in succession. It would be desirable for the plugs of 

fluid to be efficiently mixed, but to minimize the axial 

dispersion of the plug. 

FIG. 16A illustrates a plug of fluid 1602 introduced into 

the channel 1604. The channel 1604 illustrates a case 

wherein the wells of the present invention are not present 

and represents a baseline case. The mixed plug 1606 is 

shown downstream. 

FIG. 16B illustrates an embodiment of the present invention wherein a plug of fluid 1608 is introduced into a channel 

1610 in which four wells 1612, 1614, 1616, and 1618 are 

disposed. The mixed plug 1620 is shown downstream. For 

FIG. 16B, rEOM is set to 2.00. 

FIG. 17 illustrates the results of a computational analysis 

of the flow of the embodiments of FIGS. 16A and 16B. The 

curves 1702 and 1704 illustrate the average concentration of 

the plug as it passes the outlet of the mixing channel over 

time. Curve 1702 represents the plug of fluid from FIG. 16A 

and curve 1704 represents the plug of fluid from FIG. 16B, 

with rEOM equal to 2.00. 

The cross section 1706 represents the analysis results for 

the point 1708 and cross section 1710 represents the analysis 

for the point 1712. Both cross sections are the approximate 

high point of the concentration. For cross section 1706, the 

plug flow with no wells, the average concentration of the 

reagent is approximately 28% higher than the cross section 

1710. However, the standard deviation, an approximate 

measure of the degree of mixing, is approximately 3.6 times 

higher for cross section 1706 wherein no wells were present. 

The lower standard deviation of the mixture that passed 

through the inventive wells indicates that the plug of reagent 

was very well mixed. Further, from the curve 1712, the plug 

of fluid is still intact, although slightly elongated when 

compared to the reagent that was not passed over the 

inventive wells. 

The present invention is a passive device that greatly 

enhances the mixing of reagents under electrokinetic flow, 

and to a lesser degree, under pressure driven flow. The 

present invention significantly decreases the channel length 

required for mixing reagents by placing wells in the flow 

channel at oblique angles to the axis of flow. The wells may 

be of various depths, however, for a given set of reagents, 

flowrates, and channel geometries, there may be an optimum 

depth of a well wherein an increased depth may not increase 

the mixing effectiveness. 

Fabrication and operation of the passive micro mixing 

apparatus of the present invention is simplified over 

approaches that require splitting the fluid flow into multiple 

smaller channels and then recombining. By contrast, mixing 

in accordance with the present invention is the result of 

transverse flow and fluid folding in a single channel, thereby 

permitting effective mixing of confluent streams with high 

flow rates. 

Electroosmotic flow is a surface driven mechanism that 

may be enhanced to change the performance of the present 

invention. For example, increasing the electroosmotic 

mobility of selective surfaces such as the wells has been 

shown to increase the effectiveness of the mixer. 

The manufacturing process and equipment used in the 

experiments referenced in this specification are herein 

defined. 

Reagents and Materials. Laser Grade Rhodamine B was 

used as supplied by Acros Organics (Belgium) and dissolved 

in 20 mM, pH 9.4 carbonate buffer to a final concentration 

of 0.11 mM Rhodamine B. The buffer solution was made 

using deionized water from a Millipore Milli-Q system 
US 6,907,895 B2 


9 10 


(Bedford, Mass.), and was filtered before use with a syringe 

filter (pore size 0.22 ,urn). 

Microchannels were made using polycarbonate sheet (PC; 

Lexan, G E Co., Mt. Vernon, Ind.). Poly(ethylene terephthalate glycol) (PETG; Vivak, DMS Engineering Plastic 

Products, Sheffield, Mass.) was used to cover and seal the 

microchannel substrate. The glass transition temperature of 

PC and PETG are approximately 150° C. and 81°C., 

respectively. Polycarbonate was chosen as the substrate 

material because it has a high absorption cross section to 248 10 

nm light (the wavelength of the excimer laser), therefore 

ablated structures have minimal surface roughness «5 nm). 

PETG was chosen to seal the microchannels because its 

glass transition temperature is well below that of pc. 15 

Therefore, thermal sealing can be performed at a temperature that does not cause distortion of the PC microchannel. 

Hot Imprinting Method. Prior to imprinting, the PC 

substrate was blown clean with ionized air. Channels were 

hot imprinted in the substrate material using a silicon stamp 

with a trapezoidal-shaped raised T-channel. The PC was 20 

place over the silicon stamp, the two items were then placed 

between two aluminum heating blocks, and then the temperature was raised to 155° C. Next, the assembly was 

placed in a hydraulic press and a pressure of 13.8 MPa (2000 

psi) was applied for 1.5 hours. The imprinted substrate was 25 

then removed from the template and allowed to cool to room 

temperature. Channel dimensions were measured by optical 

profilometry. 

Laser Ablation Method. A 248 nm excimer laser system 30 

(LMT-4000, Potomac Photonics, Inc., Lanham, Md.) was 

llsed to ablate microstructures within the pre-formed PC 

microchannel. The excimer laser system, FIG. 18, contains 

a laser light source 1, a round aperture (200,um diameter) 2 

for delimiting the size and shape of the beam 3, a focusing 35 

lens (lOx compound) 4, a visible light source 5, a CCD 

camera to image the ablation process 6, and a controllable 

X-Y stage 7 with a vacuum chuck 8 to hold the substrate 9 

in place. Also, a nozzle 10 was present to sweep nitrogen 

over the substrate 9 during processing, and a vacuum nozzle 

11 was located on the opposite side of the stage to remove 40 

debris. For the experiments conducted here, the sizedelimiting aperture was chosen such that the ablated features 

would be smaller than the dimensions of the channel. Also, 

the X-Y stage was moved linearly at a rate of 1 mmls, and 45 

the ablated wells were at a 45° angle relative to the axis of 

the main channel. The average power level per pulse was set 

to 2.04 ,uJ+I-0.14,uJ. The frequency of pulses was set to 200 

Hz, with a constant pulse width of 7 ns. The light after being 

focused exposed a circular area of 1.90x10-6 cm2 . 50 

Measuring Well Depth and Profile. The depth of the 

ablated wells was measured by cutting the substrate with a 

microtome (Microm HM335 E, Walldorf, Germany) either 

perpendicular to the axis of the outlet channel or parallel to 

the slanted wells. The substrate was cut so that the edge of 55 

the substrate was within a few microns of the wells. The 

wells were then imaged and measured using white light 

microscopy. 

Microchannel Sealing Procedure. The pre-formed microchannels were covered and thermally sealed with a flat piece 60 

of PETG (referred to as the 'lid' throughout the rest of the 

text) of similar dimensions to the pc. Prior to bonding, the 

lid and the channel were cleaned with compressed nitrogen 

gas. The lid was then placed on top of the channel, and the 

two pieces were clamped together between microscope glass 65 

slides and bonded by heating in a circulating air over at 

90.0° C.+1-0.5° C. for 13 minutes. It is important to keep the 

time and temperature as low as possible in the sealing 

process to avoid physical alteration of the microchannel. 

For the electroosmotic flow studies, 3 mm diameter 

circular holes in the lid provided access to the channels and 

served as fluid reservoirs. For the pressure driven flow 

studies, 0.8 mm diameter circular holes in the lid, located at 

the ends of each inlet channel, provided access to insert a 

section of hollow stainless-steel tubing. A 3 mm diameter 

hole in the lid at the end of the outlet channel served as a 

waste reservoir. For each experiment, the channel arms were 

fixed to a length of 8 mm. 

Flow Image Acquisition. Flourescence imaging of the 

rhodamine dye was performed using a research fluorescence 

microscope equipped with a lOx objective, a mercury arc 

lamp, a rhodamine filer set, and a video camera (COHU, San 

Diego, Calif.). Digital images were acquired using Scion 

ImageTM software and a Scion LG-3 frame grabber (Scion, 

Inc., Frederick, Md.). For each experiment, images were 

captured every %oth of a second over a duration of 0.67 s, 

averaged, then recorded. 

Experimental Set-up. To image the mixing under electroosmotic flow, the microchannels were initially filled with 

the carbonate buffer solution. Then, an equal amount 

(typically 40 ,uL) of buffer was placed in one inlet channel 

reservoir and in the outlet channel reservoir, while the 

second inlet reservoir was filled with the rhodamine-labeled 

buffer. Platinum electrodes were then placed in contact with 

the solution in the reservoirs such that the two inlet reservoirs were fixed to ground and the potential was applied to 

the outlet channel reservoir. The microchannel was placed 

beneath the fluorescence microscope described in the previous section, and images were acquired at several different 

applied voltages (0 to -1750V), beginning with zero applied 

voltage to verify that there was minimal flow resulting from 

hydrostatic pressure. The current through the microchannel 

was determined by measuring the voltage drop across a 100 

kQ resistor (typically less than 1/1000 the resistance of the 

microchannel) connected to the high voltage supply in series 

with the microchannel. For pressure driven flow studies, a 

programmable syringe pump (Harvard Apparatus PHD 

2000, Holliston, Mass.) was interfaced to the stainless 

tubing in the inlet reservoirs via Teflon tubing. 

The foregoing description of the invention has been 

presented for purposes of illustration and description. It is 

not intended to be exhaustive or to limit the invention to the 

precise form disclosed, and other modifications and variations may be possible in light of the above teachings. The 

embodiment was chosen and described in order to best 

explain the principles of the invention and its practical 

application to thereby enable others skilled in the art to best 

utilize the invention in various embodiments and various 

modifications as are suited to the particular use contemplated. It is intended that the appended claims be construed 

to include other alternative embodiments of the invention 

except insofar as limited by the prior art. 

What is claimed is: 

1. A method of mixing two confluent laminar flows in 

microchannels comprising: 

providing a first inlet stream and a second inlet stream that 

meet at a confluence point to produce a confluent 

stream; 

passing said confluent stream through a mixing channel, 

said mixing channel comprising a plurality of substantially straight unconnected wells, said wells being 

oriented substantially longitudinally across the width of 

said mixing channel and diagonally across said mixing 

channel; and 

producing a mixed laminar flow at the output of said 

mixing channel. 
US 6,907,895 B2 


11 12 


2. The method of claim 1 wherein alternating wells are 

configured perpendicular to each other. 

3. The method of claim 1 wherein said wells are configured parallel to each other. 

4. The method of claim 1 wherein said microfiuidic 

streams are propelled by pressure. 

5. The method of claim 1 wherein said microfiuidic 

streams are propelled by electrokinetics. 

6. The method of claim 5 wherein at least a portion of the 

surface of said wells having an electroosmotic mobility that 

is different from at least a portion of the surface of said 

mixing channel. 

7. The method of claim 1 wherein said first inlet stream 

comprises a plug of reagent. 

* * * * *