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US007029561B2 

(12) United States Patent (10) Patent No.: US 7,029,561 B2 

Ross et al. 

(54) 	 FLUIDIC TEMPERATURE GRADIENT 

FOCUSING 

(75) 	 Inventors: David Ross, Silver Spring, MD (US); 

Lauric 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 tenn of this 

patent is extended or adjusted under 35 

u.s.c. 154(b) by 483 days. 

(21) App!. No.: 10/197,331 

(22) Filed: Jul. 18, 2002 

(65) Prior Publication Data 

US 2003/0019752 Al Jan. 30, 2003 

Related U.S. Application Data 

(60) 	 Provisional application No. 60/323,404, filed on Sep. 

19, 2001, provisional application No. 60/307,691, 

filed on Jul. 25, 2001. 

(51 ) 	 Int. Cl. 

GOIN 271447 (2006.01) 

GOIN 271453 (2006.01) 

(52) 	 U.S. Cl. ...................... 204/451; 204/601; 204/450; 

204/600 

(58) 	 Field of Classification Search ........ 204/451-455, 

204/601-605,450,600 

See application file for complete search history. 

(56) References Cited 

U.S. PATENT DOCUMENTS 

3,664,939 A 5/1972 Luner et al. 

5,021,646 A * 6/1991 Weinberger et al. ... 250/227.11 

5,759,370 A 6/1998 Pawliszyn 

(45) Date of Patent: 	 Apr. 18, 2006 

5,795,720 A * 8/1998 Henco et al. .................. 435/6 


6,277,258 BI * 8/2001 Ivory et al. ................. 204/450 


6,749,735 Bl * 6/2004 Le Febre .................... 204/601 


OTHER PUBLICATIONS 

Grushka et al. (Effect of Temperature Gradients on the 


Efficiency of Capillary Zone Electrophoresis Separations, 


Ana!' Chem. 1989, 61, 241-246), Feb.* 


Hinckley ("Electrophoretic Thermal Theory: I Temperature 


Gradients and their Effects," Journal of Chromatography, 


109 (1975 209-217), Jun.* 


Knox et al. (Temperature Effects in Capillary 


Electrophoresis. 1: Internal Capillary Temperature and 


Eftect upon Performance. Chromatographia vol. 38, No. 


3 / 4 Feb. 1994).* 


Birmes et a!. ("Analysis of the conformational transitions of 


proteins by temperature-gradient gel electrophoresis," 


Electrophoresis 1990, 11, 795-801), month unknown.* 


(Continued) 

Primary Examiner-Alex Noguerola 

(74) Attorney, Agent, or Firm-Stites & Harbison PLLC; 

Ross F. Hunt, Jr. 

(57) ABSTRACT 

A method and device are provided for concentrating and 

separating ionic species in solution within a fluidic device 

having a fluid conduit such as a channel or capillary. The 

concentration is achieved by balancing the electrophoretic 

velocity ofan analyte against the bulk flow of solution in the 

presence of a temperature gradient. Using an appropriate 

buffer, the temperature gradient can generate a corresponding gradient in the electrophoretic velocity so that the 

electrophoretic and bulk velocities sum to zero at a unique 

point and the analyte will be focused at that point. The 

method and device may be adapted for use with a variety of 

analytes including fluorescent dyes, amino acids, proteins, 

DNA and to concentrate a dilute analyte. 

37 Claims, 6 Drawing Sheets 

x 

- -	 - - - - - - - UEP / 

/

-------_/ 

/ 

u ................................... 
 x

UT 

•... ... .... .,', .. "........................ 
UB 
US 7,029,561 B2 

Page 2 

OTHER PUBLICATIONS 

[iu et al. ("Separation of Chlorophenols by Capillary Zone 


Electrophoresis. The influence of pH of the Electrophoretic 


Buffer on Selectivity," Short Communications 1. High 


Resol. Chromatogr., vol. 21, May 1998). * 


Grossman et al. ("Effect of Buffer pH and Peptide Compo­


sition on the Selectivity of Peptide Separations by Capillary 


Zone Electrophoresis," Analytical Biochemistry 173, 265­

270 (1988)), Sep.* 


Lochmiiller et al.; "Open-Channel Isoelectric Focusing In 


Thermally Engendered pH Gradients"; Journal 0/ Chroma­


tography, 480 (1989); pp. 293-399. 


Liang Zhu, Hian Kee Lee, Bingcheng Lin and Edward S. 


Yeung, "Spatial temperature gradient capillary 


electrophoresis for DNA mutation detection", 


Electrophoresis, 22, 3683-3687 (2001). 


Roger M. Wartell, Seyed Hosseini, Sandra Powell and Jian 


Zhu, "Detecting single base substitutions, mismatches and 


bulges in DNA by temperature gradient gel electrophoresis 


and related methods", Journal 0/ Chromatography A, 806, 


169-185 (1998). 


Chen-Wen Whang and Edward S. Yeung, "Temperature 


Programming in Capillary Zone Electrophoresis", Anal. 


Chem., 64, 502-506 (1992). 


B. Crane, C. Hogan, L. Lemlan and I.W. Hunter, "DNA 

mutation detection via fluorescence imaging in a spatial 

themlal gradient, capillary electrophoresis system", Review 

a/Scientific Instruments, vol. 72, 11,4245-4251 (2001). 

* cited by examiner 
u.s. Patent Apr. 18,2006 Sheet 1 of 6 US 7,029,561 B2 

PRIOR ART 


4 

Electrodes 

3 ~ =-I---------~ 5 

~______

V

_----A---__V __----) 

Section 1 

"'\ 

Section 2 

Microchannel 

Fig. l(a) 

4 

x 

Fig. 1(b) 
u.s. Patent Apr. 18,2006 Sheet 2 of 6 US 7,029,561 B2 

PRIOR ART 

--------,,

,


, 

- - - - - - - - - UEP 

u(z) 1------~----_3to "--____z_ LIT 

, •••••••••••••••••••••••••••••••••• 1, ••••••••••••••• ,............... 
Uf3 

Fig. 2 
u.s. Patent Apr. 18,2006 Sheet 3 of 6 US 7,029,561 B2 

1. 	 1214 	 hot 	 cold ~ . Fig.3(a) 	 . high

-L Ir-.:...._-_-=-__.;..;,;,;,,;;,;~~ microchannel ________.. voltage

\ .. 10 

1 ____'" 

Fig. 3(b) T(z) 	 ~ '-----­

z 

Fig. 3 (c) f(7(z» --------/ 


z 

Un •••••••• '11 ................., •••• " ..... , ............... _••• It ....................... 


Fig. 3 (d) u(z) I--______~-___	--:;;. 

z 

_... _-_ ...... - ..... 

'\ 

, 

\ 

,-----_ ..... - ..... ­
u.s. Patent Apr. 18,2006 Sheet 4 of 6 US 7,029,561 B2 

29 

V2 

24 22 

~ 27 

20 

Fig.4(a) 

) 

x 

Fig.4(b) 

f(T) 

Fig. 4 (c) 

x 

- - - - - - - - - UEP 

/

-------_/ 

/ 

/ 

U~--------~--------__7 

.... .........................................'--___X_ UT 


................................. Ua 


Fig. 4 Cd) 
u.s. Patent Apr. 18,2006 Sheet 5 of 6 US 7,029,561 B2 

55
 61 


54 


51 


Fig. 5 

u.s. Patent Apr. 18,2006 Sheet 6 of 6 US 7,029,561 B2 

70 80 ___ r 
 ~ detector 
 '--___--I 

74 


77 


77 


Fig. 6 

10 

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30 

40 

50 

60 

US 7,029,561 B2 


1 

FLUIDIC TEMPERATURE GRADIENT 

FOCUSING 

CROSS-REFERENCE TO RELATED 

APPLICATIONS 

This application claims benefit of the filing date of both 

copending Provisional Patent Application Nos. 60/307,691, 

filed on Jul. 25, 2001, and 60/323,404, filed on Sep. 19, 

2001. 

STATEMENT REGARDING FEDERALLY 

SPONSORED RESEARCH 

This invention was made by employees of the United 15 

States Government and may be manufactured and used by or 

for the Govenunent for governmental purposes without the 

payment of any royalties. 

FIELD OF THE INVENTION 

The present invention relates to a method for electrokinetic focusing of samples, and in particular, methods for 

electro-focusing samples in fluidic devices using electric 

field gradients. 25 

BACKGROUND OF THE INVENTION 

Over the past decade a great deal of research has been 

focused on the development of teclmology related to micrototal-analytical systems. This technology is based on the 

concept of a series of microfluidic chatmels also known as 

microchatmels for the movement, separation, reaction, and! 

or detection of various chemicals or biological compounds 

such as amino acids, proteins, atld DNA. 

35 One disadvantage with prior micro fluidic devices is that 

there is frequently a mismatch between the extremely small 

quatltities of sample used for analysis atld the often much 

larger quatltities needed for loading the sample into the 

microfluidic device atld transporting the sample to the point 

of analysis. For example, a typical analysis sample may be 

around one nanoliter or less of a liquid containing sample 

that is injected into a separation chatmel and then separated 

electrokinetic ally as it moves down the chatmel to a detection region. However, the channels used to transport the 45 

sample to the injection point are typically also filled with the 

Satllple, thus increasing the required amount of the sample 

by a factor of 100 or more. In addition, the sample is 

typically loaded onto the micro fluidic device into a reservoir 

from a pipette so that in all, approximately 99.9% of the 

sample is discarded as waste. 

Electric field gradient focusing is one way of addressing 

the problem of requiring a large sample for atlalysis due to 

the inefficiencies of conventional devices which result in 

wasted sample. Electric field gradient focusing can be used 55 

to concentrate samples at a given point within a micro fluidic 

device before the analysis step. Further, the electric field 

gradient can be used to concentrate all of the sample at the 

begiuning of the separation chatmel so that very little of the 

sample would be wasted. 

Electric field gradient focusing is accomplished by the 

application of an electric field gradient within a microchannel. In response to the electric field gradient, there is a 

corresponding gradient in the electrophoretic velocity of any 

ion within the microchatmel. The total velocity of the ion is 65 

the sum of its electrophoretic velocity atld the bulk fluid 

velocity. If these two components of the velocity are in 

2 

opposite directions, they can be balanced so that the molecule will have zero total velocity. 

When there is a gradient in the electrophoretic velocity, 

the balance between bulk and electrokinetic velocities can 

occur at a single point within the microcha11l1el and therefore 

can result in focusing of ions at that point. Typically, the 

electric field gradient used in focusing is generated by the 

external manipulation of the electric field in the middle of 

the microcha11l1el through the use of conducting wires, salt 

bridges, porous membranes, or other structures that will pass 

electric current but will restrict the flow of bulk fluid and 

analytes that are to be focused. 

Several recent developments with regard to focusing 

methods in microfluidics, and in particular, the use of 

electric field gradients, have been made. A description of 

related methods of focusing can be found in C. F. Ivory, W. 

S. Koegler, R. L. Greenlee, and V. Surdigio, Abstracts of 

Papers of the Americatl Chemical Society 207, 177-BTEC 

(1994); c. F. Ivory, Separation Science and Teclmology 35, 

1777 (2000); Z. Huang and C. F. Ivory, Analytical Chemistry 

71, 1628 (1999); W. S. Koegler and C. F. Ivory, Journal of 

Chromatography a 726, 229 (1996); and P. II. Ofarrell, 

Science 227, 1586 (1985), all of which are hereby incorporated by reference. 

To illustrate the basic principles disclosed in these publications, reference is made to FIG. lea) which depicts a 

length of buffer-filled microcha11l1el of constant cross-sectional area with an electrode, denoted 4, in the middle, and 

two further electrodes at each end, denoted 3 and 5, so that 

the voltages Vj' V3 at the ends and the voltage at the 

middle of the chatmel can be controlled. A single species of 

negatively charged analyte is present in a buffer that is 

provided to the microcha11l1el. The electrical comlection, 

represented as electrode 4, can be accomplished with a 

simple metal wire as depicted in FIG. lea), or through a 

more complicated structure consisting of additional fluid 

chatmels and porous membrane structures or salt bridges. 

The electric field in the section 1, i.e., the cha11l1el between 

electrodes 3 and 4 is Ej=(V2-Vj)/(l/2) and the electric field 

in section 2, i.e., between electrodes 4 and 5, is E2=(V3 ­

V2)/(l/2), where Vjl V2, and V3 are the voltages applied to 

the three electrodes 3, 4, and 5, and I is the length of the 

microchatmel. If, Ej differs trom E2 as shown in FIG. l(b), 

the electrophoretic velocity of the analyte in the cha11l1el, 

uEF, will be different in section 1 than in section 2. If an 

overall bulk fluid velocity, uB<O, is applied, e.g., either 

electro-osmotic or pressure-driven, the bulk fluid velocity 

must be the same, due to continuity, in all parts of the 

microchatmel. The total velocity of the analyte, ur=UB+UEP' 

will then be the sum of the electrophoretic and bulk velocities, which can differ in section 1 from section 2. 

The use of the microchannel device of FIG. lea) for 

focusing of the ions is illustrated in FIG. 2 where UT j>O>uT 

2, so that the ions flow into the middle from both directions 

and are thus focused in the middle of the cha11l1el near 

electrode 4. 

One major drawback to electric field gradient focusing is 

that the microchatmel device tends to be difficult to construct 

and that it requires the control of voltage on an additional 

electrode, e.g. 4 of FIG. lea), that is used to apply the 

electric field gradient. In addition, if electrodes are used to 

generate electric field gradients, unwanted chemical products will be generated electrochemically at the buffer-electrode interface. If the electric field gradient is produced 

through the use of a salt bridge or membrane, the electro­
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US 7,029,561 B2 


3 4 


chemical products can be avoided, however only chemical 

species that cannot pass through the membrane or salt bridge 

can be focused. 

Two additional methods for concentrating a sample 

include sample stacking and field amplified sample injection 

in which a sample is concentrated as the sample crosses a 

boundary between low and high conductivity buffers. These 

methods can achieve preconcentration factors of 100 to 

1000-fold although these methods require multiple buffers. 

Sweeping is yet another concentration method which is 

capable of a very high degree of sample concentration (e.g" 

up to 5000-fold), but is useful only for small hydrophoic 

analytes with a high affinity for a mobile micellular phase. 

An additional teclmique for concentrating an ionic sanlple 

includes isoelectric focusing. Isoelectric focusing is com­ 15 

monly used for the concentration and separation of proteins 

and involves the focusing of analytes at their respective 

isoelectric points (pIs) along a pH gradient. 

Two examples of recent isoelectric focusing techniques 

are provided by U.S. Pat. No. 3,664,939 to Luner et al. and 

U.S. Pat. No. 5,759,370 to Pawliszyn. Both references relate 

to isoelectric focusing with pH gradients that are created by 

the application of a temperature gradient. The isoelectric 

focusing uses a pH gradient to focus analytes and in particular proteins, at their isoelectric points. The isoelectric 25 

point is the pH at which the analyte has zero electrophoretic 

mobility, i.e., approximately zero charge. pH gradients for 

isoelectric focusing are typically generated using ampholyte 

mixtures or illllllObilized ampholytes in gels. The two above 

referenced patents are included here as examples of prior art 

uses of temperature gradients for focusing. It is actually very 

unusual for isoelectric focusing to be done with a pH 

gradient generated with using a temperature gradient. 

One disadvantage with isoelectric focusing is that it is 

limited in application because it can only be used with 35 

analytes with an accessible pI. Additionally, the concentration to which a protein can be focused with isoelectric 

focusing is severely limited due to the low solubility of most 

proteins at their pIs. 

BRIEF SUMMARY OF THE INVENTION 

The present invention concerns a method and device for 

concentrating and separating ionic species in solution within 

fluid conduits which include chmmels, microchaunels, and 45 

capillary tubes. The concentration is achieved by balancing 

the electrophoretic velocity of an analyte against the bulk 

flow of solution in the presence of a temperature gradient. 

Using an appropriate buffer, the temperature gradient can 

generate a corresponding gradient in the electrophoretic 

velocity so that the electrophoretic and bulk velocities sum 

to zero at a unique point and the analyte will be focused at 

that point. The present invention may be adapted for use 

with any charged analyte, including fluorescent dyes, amino 

acids, proteins, DNA, cells, and particles and may provide 55 

up to or, in some instances, exceed a 10000-fold concentration of a dilute analyte. 

One aspect of the present invention concerns a method for 

directing ionic analytes contained in an ionic buffer solution 

of a system and which may include concentrating or separating analytes present in the buffer solution. The method 

includes producing an electric current flow in an ionic buffer 

solution containing at least one species of ionic analyte to 

cause the analyte ions to migrate electrophoretic ally. A 

temperature gradient is established in the buffer solution to 65 

have a significant component substantially aligned with the 

current flow, to thereby generating a gradient of the electrophoretic velocity of the analytes. A bulk flow is produced 

in the buffer solution such that the bulk flow has a significant 

component substantially aligned in the direction opposite the 

direction of the electrophoretic migration of one or more of 

the analytes so that the total velocity of one or more of the 

analytes is equal to zero at some point in the system. 

According to another aspect of the present invention, a 

fluidic device includes a fluid conduit and an ionic buffer 

disposed in the conduit. At least one source or sink of heat, 

thennally coupled to the fluid conduit, is provided for 

establishing a temperature gradient having a significant 

component substantially aligned with the current flow so as 

to fonn an electrophoretic velocity gradient within the fluid 

conduit. A voltage potential source is provided for applying 

an electric field along a length of the fluid conduit and a 

current source provides an electric current flow through the 

ionic buffer in the fluid conduit. A source of bulk fluid flow 

provides for an opposing flow of the buffer in the fluid 

conduit. In alternate, further embodiments, the ionic buffer 

has either a temperature dependent ionic strength or a 

temperature dependent pH such that when a temperature 

gradient is applied to the fluid conduit, an electrophoretic 

velocity gradient is established in the ionic buffer present in 

the fluid conduit. 

One advantage or feature of the present invention is 

provided by a technique that allows for simultaneous concentration and separation in a mauner similar to isoelectric 

focusing but which is adoptable for use with any charged 

analyte and is not limited to molecules for a specific pI or 

range of pIs. Further, the temperature gradient focusing of 

the present invention can be used to achieve higher degrees 

of salllple concentration, e.g., more than 10,000 fold concentration of a dilute sample, when compared with any prior 

single sample preconcentration method. 

A further feature of the present invention is that the 

electrophoretic velocity gradient is formed within the channel or capillary in response to the temperature gradient 

without the need for externally manipulated voltages or 

complicated and difficult to fabricate semi-permeable structures. 

Further features and advantages of the present invention 

will be set forth in, or apparent from, the detailed description 

of preferred embodiments thereof which follows. 

BRIEF DESCRIPTION OF THE DRAWINGS 

The invention will now be described in detail with respect 

to preferred embodiments with reference to the accompanying drawings, wherein: 

FIG. lea) is a schematic depicting a prior art micro channel device which provides for electric field gradient and FIG. 

l(b) is a plot of the electric field versus distance (x) along the 

microchannel of FIG. lea); 

FIG. 2 is a plot of velocity versus distance along the 

microchallllel of FIG. lea); 

FIG. 3(a) is a schematic illustration of temperature gradient focusing and fluid conduit in the form of a micro channel in accordance with the present invention, FIG. 3(b) 

depicts temperature distribution along the microchannel of 

FIG. 3(a), and FIG. 3(c) is a plot of the function 

0-(20)· ry(20) 

j(T) o-(T). 'I(T) 
10 

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30 

40 

50 

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US 7,029,561 B2 


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plotted as a function of the distance along the microchmmel 

of FIG. 3(a), and FIG. 3(d) is a plot depicting velocity as a 

function of distance along the microchannel; 

FIG. 4(a) is a schematic illustration of a microchmlllel for 

temperature gradient focusing created by louIe heating 

according to another embodiment of the present invention, 

FIG. 4(b) depicts the temperature profile along a length of 

the microchannel of FIG. 4(a), FIG. 4(c) depicts the electric 

field profile along a length of the microchannel of FIG. 4(a), 

and FIG. 4(d) is a plot showing electrophoretic velocity, bulk 

velocity, and total velocity vs. distance along the microchannel of FIG. 4(a); 

FIG. 5 is a schematic drawing of a fluidic device according to fhrther embodiment of the present invention; and 

FIG. 6 is a schematic drawing of a capillary fluidic device 15 

according to an alternate embodiment of the present invention. 

DETAILED DESCRIPTION OF THE 

INVENTION 

The present invention provides temperature gradient 

focusing of a sample in a fluidic device which includes a 

fluid conduit such as a channel or capillary tube. Temperature gradient focusing focuses analytes by balancing the 25 

electrophoretic velocity of an analyte against the bulk velocity of the buffer containing the analyte. If there is an 

appropriate gradient in the electric field, the total velocity of 

a given charged analyte, as determined by the sum of the 

bulk and electrophoretic velocities, can be set to zero at any 

point along the channel and all the analyte in the system is 

moved toward that point. However, in contrast to electric 

field gradient focusing where the electric field gradient is 

applied using a combination of electrodes and membranes, 

using temperature gradient focusing of the present inven­ 35 

tion, the necessary electric field gradient is produced by the 

application of a temperature gradient. 

Further description of the present invention will now be 

made with reference to the drawings, and in particular to 

FIG. 3(a), where a buffer-filled microchannel 10 includes 

electrode cOllllections 12. 14 at each end. The velocity of an 

analyte in the microchmmel 10 is given by the sum of its 

electrophoretic velocity, UEP' m1d the bulk velocity, UB , of 

the buffer: 45 

If there is a gradient in the electrophoretic velocity, the 

bulk velocity can be adjusted so that the total velocity is 

equal to zero at a single point along the channel, and the 

analyte will be focused at that point. The electrophoretic 

velocity of an analyte in the microchannel 10 is given by the 

product of the electric field. E, and the electrophoretic 

mobility of the analyte: uEP=E-flEP' 

A temperature gradient is applied along the length of the 

channel as shown in FIG. 3(b). This results in corresponding 55 

gradients in both the electric field E and the electrophoretic 

mobility ~lEP' 

The electric field in the microchannel 10 is given by: 

I 

E=-, A·CT 

where I is the electric current running through the micro- 65 

channel 10, A is the channel cross-sectional area of the 

microchannel 10, m1d 0 is the conductivity of the buffer. 

Since the conductivity of the buffer is temperature-dependent, the electric field is also temperature-dependent. Here, 

constant current is presumed because the current running 

through any given section of the microchmlllell 0 will be the 

san1e for all parts of the microchmmel, whereas the voltage 

drop across a portion of the microchannel 10 and the electric 

field in the microchannel 10 will depend on the temperature 

of that portion. One skilled in the art will readily appreciate 

that the present temperature gradient focusing differs from 

electric field gradient focusing in that in electric field 

gradient focusing, the velocity gradient that is used for 

focusing results from a gradient in the electric field imposed 

by the addition or subtraction or current from point or points 

within the microchannel. 

Using microchannel 10, it is possible to manipulate the 

conductivity of the buffer by changing the temperature. 

Consequently, it is possible to produce electric field gradients in microfluidic devices, such as microchannel 10, 

through the application of a temperature gradient. 

At fixed current density, the electric field in microchmlllel 

10 is inversely proportional to the conductivity of the buffer 

solution in the microchannel. Most often. the primary temperature dependence of the conductivity is due to the variation of the solvent viscosity with temperature, so it can be 

written as a=00'Y](20)/(Y](T)J(T)), where 0 is the conductivity, 00 is a constant, Y](T) is the temperature dependent 

viscosity, and f(T) is a fi.mction that accounts for any other 

temperature dependence. Similarly, the temperature dependence of the electric field is given by E=Eo'Y] (T)J(T)/Y] (20). 

where E is the electric field and Eo is a constant. 

For most buJlers, the function f(T) is constm1t or only 

weakly dependent on temperature. However, it cm1 be nonconstant. i.e., variable, if. for exmnple, the ionic strength of 

the buffer is temperature dependent. Advantageously, the 

buffers of the present invention are characterized by a 

non-constant f(T). 

The electrophoretic mobility of an ionic (e.g., analyte) 

species in the buffer is also dependent on the viscosity, and 

so can be written as ~tEp=~to'Y](20)/(Y](T)JEAT)), where flo 

and fEP(T) are defined in analogy to 00and f(T) where, for 

most analytes, f EP(T) will be constm1t. The electrophoretic 

velocity of the analyte can then be written as uEP=Eo'flo'i 

(T)/fEAT). It should be noted that if f(T) and f EP(T) have 

the same temperature dependence, e.g., they are both constant, then UEP will not be temperature dependent, m1d 311 

electric field gradient produced in this way can not be used 

for focusing. 

If, on the other hm1d, f(T) and f EP(T) do not have the 

smne temperature dependence, then temperature gradients 

will result in gradients in the electrophoretic velocity, which 

can be used for focusing as described above. 

One skilled in the art will readily appreciate a major 

advantage of this present method over some other methods 

of preconcentration is that the concentration of the buffer 

salts is completely unaffected by the focusing. This results 

from the fact that if the buffer salt is considered as an 

analyte, then, by definition, f EAT)=f(T) and there is no 

gradient in the electrophoretic velocities of the buffer salts. 

Most c0ll1l110nly this technique would be implemented 

with a buffer characterized by a strongly temperature dependent f(T) and with analytes characterized by a constm1t or 

nearly constant fEP(T). However, the present temperature 

gradient focusing can also be implemented in a system in 

which f(T) is constant and fEP(T) is not, or in which both 

f(T) and fEAT) are non-constm1t, but differ in their temperature dependence. 
10 

20 

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TIle counterbalancing bulk flow can be applied electroos­ about 500 ~ll. The depth of all portions of the chailllel was 

motically if the electro-osmotic mobility does not differ too about 30~. The total length ofthe microchallnel was about 

much from the electrophoretic mobility of the analyte. If the 2 cm, with the length of the section 28 divided by the total 

electro-osmotic mobility is written as ~lEo=/lEoo'11(20)hl(T), length, xssO.8. Access to each end of the microchaunel was 

then by adjusting the ratio of the lengths of the hot and cold provided by a 3 nllll hole through the lid piece of the 

chaunels, (assuming f EAT)=constant) focusing can be microchannel. 

achieved if f(cold)lf(hot)<-/lo/~LEoo<f(hot)/f(cold), where An 8 ~Lmol/L solution of carboxyfluorescein in the 1.8 M 

f(hot»f(cold). If f(hot)<f(cold), then the inequalities have Trisiboric buffer was prepared. The analyte to be concenthe opposite sign. If x is the fraction of the total chailllel trated was the carboxyfluorescein. Detection of the analyte 

length that is hot then focusing will occur if: xJ(hot)lf was performed using a fluorescence microscope and CCD 

(cold)+(l-x)<-/lol/lEo0 <x+(I-x)J(cold)lf(hot), where cameras. Simultaneous color and grayscale images were 

f(hot»f(cold). By adjusting x, it is then possible to tune the obtained. 

range of analyte mobilities that are focused. To demonstrate gradient focusing using louIe heating, the 

It should be noted that this can also be done for micro- microcharmel was filled with the carboxyfluorescein soluchailllels of non-constant cross-section. The final results are 15 tion and 1900 V was applied along its length, with the 

essentially unchanged, since in most instances, the depen- positive voltage V2 applied to the narrow end via electrode 

dence on the cross-sectional area of the chailllel cancels out 22, and the wide end held at ground at electrode 24. 

in the equations. As a result, it is possible to generate the After 6 min., the carboxyfluorescein was highly concentemperature gradient using louIe heating within the micro- trated at the junction between sections 26 and 28 of the 

chaunel. This would serve to simplify the design and opera- microchannel 20. The concentration factor achieved by 

tion of a microfluidic device using this technique even using this example was typically about 100-fold per minute. 

further, since the focusing and the temperature gradient Referring now to FIG. 5, in order to have better control of 

could be produced using the same pair of electrodes as the temperature gradient, experiments were done using three 

illustrated in FIG. 4(a). temperature zones, two cold zones provided by cooling 

Microcharmel 20 shown schematically in FIG. 4(a), has 25 copper blocks 53a, 53b covering much of the ends of the 

electrodes, 22, 24, and two sections, sections 26, 28, of microcharmel 50, and one hot zone provided by heated 

different cross-sectional area. Section 26 has a cross-sec- copper block 52. The microchailllel50 was made out of thin 

tional area of 27 and section 28 has a cross-sectional area of (125 ~) sheets ofpoly( carbonate) substrate 51, which were 

29. The electrical resistance per unit length of each section pressed onto the copper blocks 52, 53a, 53b. Thermal 

is given by: R1=1I(0'A1), where 0 is the conductivity of the contact between the poly(carbonate) and the copper blocks 

buffer in the microcharmel 20. When a current, I, is passed was insured using a thermally conductive adhesive 56. The 

through the microcharmel 20, the power per unit length copper blocks 52, 53a, 53b were arranged so that there was 

dissipated through louIe heating in each section will be: a 1 l1lln gap 58 between the heated copper block 52 and the 

P]=I2

·RI=I2/(oAI). In general, the resulting temperature in cooling copper block 53a and a 2llllll gap 59 between heated 

section 28 will be higher than that in section 26, as shown 35 copper block 52 and the cooling copper block 53b. 

in FIG. 4(b): T2>TI' The electric field in each section of the Microchailllel 50 also includes electrodes 55, 54, buffer 

microcharmel 20 is given by the current multiplied by the reservoirs 57, arld a narrow hot zone 50a near the middle of 

resistance per unit length: Ei=I'Ri=II(oAi)=hl (T}f(T1)/ the microchamlel50. The heated copper block 52 was heated 

(00 'h(20)-AJ, using a small high-power resistor embedded into the copper 

TIle electrophoretic velocity of arl analyte in each section and its temperature was regulated using a PID temperature 

of the chailllel is: UEP'=IloJ(T)])-I1(ooJEP(T)-A]). Ifa bulk controller (Omega Engineering Inc, Stamford, COilll.). To 

flow velocity is applied along the chauneL it will not be the regulate the temperature of the cold zones, 1/4 inch diameter 

sarne in each section, but will instead be given by UBi=UB 

0

I holes were drilled through the cooling copper blocks 53a, 

Ai, where, UB 

0 is a constant. The ratio of the electrophoretic 53b and cold water from a thermostatted bath (Neslab, 

velocity to the bulk velocity is then given by: u/atio=~loJ 45 Portsmouth, N.H.) was passed through them. 

(T}I1(ooJEP(T}uBo)-uorati°J(T;)/fEE,(T) via adjusting uB O Thin polycarbonate microchailllel chips, i.e. substrate 51 

so that lu/ati°I>I>lu/ati°1 as shown in FIG. 4(d), which can was attached to the copper blocks 52, 53a, 53b using 

result in focusing. Because the ratio of the electrophoretic thermally conductive adhesive 56 in the fonn of trarlsfer 

velocity to the bulk velocity does not depend on the cross- tape (3M). TIle substrate 51 was pressed against the copper 

sectional areas of the two sections, the same considerations blocks 52, 53a, 53b from above with 3 l1lln thick PDMS 

as above apply if bulk flow is applied electrooslllotically. (Sylgard 184, Dow Corning, Midland, Mich.) gaskets 60 and 

One preferred buffer system is composed of 0.9 mol/L a 2 l1lln thick acrylic (Acrylite OP-4, Cyro Industries, Mt. 

Trizma base and 0.9 mol/L boric acid in water (1.8 M Arlington, N.J.)) top plate 61, which was secured to the outer 

Trisiboric), with an expected pH of about 8.7 (at room copper clocks using nylon screws (not shown). 

temperature). From measurements ofthe conductivity of the 55 During temperature gradient focusing, a voltage potential 

buffer, the function f(T) was determined to vary from 1 at is applied to electrode 55 and electrode 54 is set to ground 

20° C. to 0.77 at 70° C. to allow microcharmel50 to provide focusing and separation 

louIe heating may be used to generate the temperature of different types of arlalytes: small dye molecules, amino 

gradient in the microcharmel device of FIG. 4(a). The acids, proteins, DNA, colloidal particles, and cells. 

following is a non-limiting example demonstrating louIe The microchamlel 50 may be fonned by imprinting with 

heating of a microchailllel of the type shown in FIG. 4(a). a micro machined silicon template and then sealed with a 

The microchallllel used for this demonstration was similar similar material according to the method disclosed in Ross, 

to the one shown schematically in FIG. 4(a). The width, i.e., D.; Gaitan, M.; Locascio, L. E., Analytical Chemistry 2001, 

cross sectional area 29, of the narrow charmel, i.e., section 73, 4117~23, herein incorporated by reference. 

28, was about 70 /lm, and the width of the wide section, i.e., 65 The copper block arrangement was also used to determine 

section 26, was of the cross sectional area 26 was about 350 the degree offocusing that could ultimately be reached with 

~. The length of the tapered portion of the chamlel was temperature gradient focusing. Begimung with a 8 nM 
10 

20 

30 

40 

50 

60 

US 7,029,561 B2 


9 10 


solution of Oregon Green 488 carboxylic acid in 1.8 M 

Trislboric, 100 min offocusing resulted in a focused plug of 

Oregon Green 488 carboxylic acid with a peak concentration 

over 80 ~lM~a greater than 10000-fold increase in concentration. 

It will become readily apparent to one of ordinary skill in 

the art that the present method provides for use innumerous 

applications. For example, temperature gradient focusing 

could be used as a preconcentration step before an analysis 

or separation or as a simultaneous concentration and separation technique. 

In addition, temperature gradient focusing may be used 

with any charged species in solution and not just small 

molecules. For exanlple, the analytes may include larger 

molecules such as proteins and DNA, or even particles and 15 

cells. Further, the present method can be used with particles 

to create packed beds of particles or cells for use in other 

analysis steps. In addition, the present method can be 

adapted for use to sort particles or cells by electrophoretic 

mobility. 

In one separation mode, the bulk velocity could be 

ramped over time to scan focused sample peaks past a fixed 

detector, e.g. the detector shown in FIG. 5. This would 

produce results similar to capillary electrophoresis but the 

widths of the sample peaks would be determined by the 25 

applied gradients and the peak heights would be determined 

by how long a given peak was in the focusing "window". If 

the ramp speed were halved, the peak heights would all be 

doubled, so that the ramp rate could be chosen dependent on 

the concentration limit of detection necessary. Alternatively, 

the focusing window could remain fixed and a scanning or 

imaging detector could be used to locate the separate peaks. 

In a further embodiment, the method may be adapted for 

a system where temperature dependence is due to something 

other than the ionic strength. An example is a system having 35 

f(T) constant but fEAT) not constant, or variable. One way 

to accomplish this would be to use a buffer with a temperature dependent pH. In such a system, this embodiment of the 

present invention is similar to isoelectric focusing schemes. 

However, the present environment dijJers from isoelectric 

focusing in that, in the present system, an opposing buffer 

flow is applied so that analytes are focused at a pH other than 

their isoelectric points. 

When using any of the embodiments of the present 

method, operating parameters which include voltage, bulk 45 

flow rate, and temperature ofthe different zones may be held 

constant with time or varied with time to affect the position 

and width of focused sample peaks. Varying of parameters 

may be accomplished using any of a number of methods 

which include the methods previously described above in 

which the focused sample peaks are scanned past a fixed 

detector. 

Advantageously, in order to achieve the fastest accumulation of analyte in the focused peak, the highest possible 

voltage should be used. However, a higher applied voltage 55 

requires a faster bulk flow which results in greater dispersion, i.e., wider focused peaks, which is disadvantageous for 

separation and for achieving preconcentration of a sample to 

a high concentration in a very narrow peak. Therefore, a 

high voltage and fast bulk flow could be used for the initial 

accumulation of analyte into a relatively broad peak, and the 

voltage flow and flow rate could be reduced to the point at 

which the peak is narrowest. Further, temperature gradients 

could be turned on and off to first concentrate the sample and 

then release the focused peak and allow it to flow on down 65 

the channel. Further, the temperature gradient can be 

adjusted to be linear or nonlinear, and the temperature 

gradient may be monotonic or non-monotonic. Thus, operating parameters may be adjusted to achieve the desired 

results. 

While the previously disclosed embodiments are directed 

to a micro channel or micro fluid device, the present method 

may be adapted for incorporation for use with substantially 

larger channels which may include millimeter and centimeter if not larger in dimension which should now be apparent 

to one of ordinary skill in the art. Because temperature 

gradient focusing uses low conductivity buffers, one can 

adapt the present method for use in much larger scale 

geometries than the micron-sized channels and capillaries 

described in detail herein. 

Further, the previously described method can be adapted 

for use in modified capillary fluidic systems known to one of 

ordinary skill in the art. FIG. 6 depicts a capillary fluidic 

system having a capillary tube 70 spatming between two 

buffer reservoirs 77. Two temperature blocks, denoted as 

heated block 72 and cooling block 73 are located along the 

length of the capillary tube 70 to provide a desired temperature gradient in the capillary tube 70. Alternatively, temperature blocks being both cooling, both heated, both at 

ambient temperature, or any combination, thereof, may be 

substituted to provide the desired temperature gradient. 

The buffer reservoirs 77 contain a buffer with temperature 

dependent ionic strength. Electrodes 74, 75 are connected at 

one end to a power supply and on the other end, are in 

contact with the bufier solution in the buffer reservoirs 77. 

The power supply applies a driving voltage through the 

capillary tube 70. A source of bulk flow is driven either by 

electro-osmosis with the applied driving voltage, by a pressure gradient applied, e.g. by a pump, or a combination of 

the two. Detector 80 is used to detect atlalytes present in the 

buffer solution. 

One of ordinary skill in the art now will readily appreciate 

that the present temperature gradient focusing differs from 

prior art methods such as sample stacking and isotachophoresis. In both cases, satnples are focused or concentrated as a result of gradients in their electrophoretic velocities. In sample stacking and isotachophoresis, the velocity 

gradients are generated at the interfaces between bufiers of 

different composition, and the point at which the concentration or focusing occurs is not stationary, but moves along 

with the electroosmotic flow in the chatmel or capillary. In 

contrast to both sample stacking and isotachophoresis, the 

velocity gradients that produce analyte focusing in the 

present temperature gradient focusing result from applied 

temperature gradients. 

Further, one skilled in the art will recognized that the 

present temperature gradient focusing differs from isoelectric focusing techniques such as those disclosed in u.s. Pat. 

Nos. 3,664,939 and 5,759,370. Unlike isoelectric focusing 

techniques in which the pH gradient is established by using 

a buffer system that has a temperature dependent pH, the 

present temperature gradient focusing utilizes a buffer that 

has a temperature dependent ionic strength. When a temperature gradient and a voltage are applied to a microchannel, the ionic strength gradient of the buffer gives rise to a 

velocity gradient, which is used for focusing. As a result, atl 

analyte present in the buffer is focused at a point where the 

analyte's total velocity, i.e., the sum of the electrophoretic 

velocity and the bulk velocity of the buffer is zero. Therefore, in the present temperature gradient focusing, the pH 

and the isoelectric point of the analyte are not critical. 

It will now be apparent to one of ordinary skill in the art 

that the present microfluidic device and temperature gradient 

focusing method provide numerous adVatltages over prior 
US 7,029,

11 	

devices and methods. The present device and method are 

simpler to implement as no imbedded electrodes or salt 

bridges are necessary. In addition, like isoelectric focusing, 

temperature gradient focusing can be used to both concentrate and separate analytes, but without the disadvantages 

associated with isoelectric focusing. 

A fiJrther advantage of the present invention is provided 

in that only a single, continuous buffer system is required. 

Solid phase extraction and related preconcentrationmethods 

of the prior art require multiple buffers where one buffer is 10 

used to carry the analyte to the preconcentrator and a second 

buffer is used to release the analyte from the preconcentrator. 

Further examples of multiple buffer systems include sample 

stacking, field amplified injection, iosotachophoresis, and 

sweeping. 15 

Further, the present temperature gradient focusing provides enlIanced concentration when compared with the prior 

art of other single preconcentration methods. 

Although the invention has been described above in 

relation to preferred embodiments thereof, it will be under­ 20 

stood by those skilled in the art that variations and modifications can be effected in these preferred embodiments 

without departing from the scope and spirit of the invention. 

What is claimed is: 

1. A method for directing ionic analytes contained in an 25 

ionic buffer solution, said ionic buffer solution having a 

temperature dependent property selected from the group 

consisting of ionic strength and pH, said method comprising 

the steps of: 

applying an electric field to an ionic buffer solution 30 

containing at least one species of ionic analyte to cause 

the analyte ions to have electrophoretic motion; 

establishing, in said buffer solution, a temperature gradient having a significant component substantially 

aligned with the electrophoretic motion of the analyte 35 

ions, which produces a gradient in the ionic strength or 

pH of said buffer solution, thereby generating a gradient of the electrophoretic velocity of the aualytes; and 

producing a bulk flow of said buffer solution to have a 

significant component substantially aligned in the 40 

direction opposite the direction of the electrophoretic 

motion of one or more of the analytes so that at least 

one of said one or more analytes will accmnulate or be 

focused at at least one point along said temperature 


gradient, the pH at said at least one point being unequal 45 

to the isoelectric point of said at least one of said one 

or more analytes that are focused at said at least one 

point. 

2. The method of claim 1, wherein said temperature 

dependent property is ionic strength and said temperature 50 

gradient establishes a gradient in the ionic strength of said 

ionic buffer solution. 

3. 	 The method of claim 2, wherein said temperature 


gradient is applied so as to produce an electrophoretic 

velocity gradient which concentrates analytes present in the 55 

ionic buffer solution. 

4. The method of claim 2, wherein said temperature 

gradient is applied so as to produce gradients in the electrophoretic velocities of the analytes present in the ionic 

buffer solution thereby causing different analytes to focus at 60 

different points within the ionic buffer solution so as to 

separate the different analytes. 

5. The method of claim 2, wherein the analyte is selected 

from the group consisting of small ions, amino acids, DNA, 

particles, cells and proteins. 65 

6. The method of claim 2, wherein the bulk flow is 

generated by electroosmosis. 

561 B2 

12 

7. The method of claim 2, wherein the bulk flow is 

generated by pressure gradients. 

8. The method of claim 2, wherein the bulk flow is 

generated by a combination of electroosmosis and pressure 

gradients. 

9. The method ofclaim 2, wherein at least one operational 

parameter selected from the group consisting of temperaure, electric field and bulk flow rate is varied over time to 

affect the position and width of focused sample peaks. 

10. The method of claim 2, wherein operational parameters consisting of temperature, electric field, and bulk flow 

ate are held constant. 


11. The method of claim 2, wherein the temperature 


gradient is one of linear and non-linear. 


12. The method of claim 2, wherein the temperature 

gradient is one of monotonic and non-monotonic. 

13. The method of claim 2, wherein the step of establishing a temperature gradient comprises applying an electric 

current to the ionic buffer solution to produce the temperaure gradient by Joule heating. 

14. The method of claim 2, wherein the ionic buffer 

solution is supplied as a continuous single buffer flow. 

15. The method of claim 2, wherein the ionic buffer 

solution and analytes are contained within a microchatmel. 

16. The method of claim 15, wherein the step of establishing a temperature gradient comprises supplying thermal 

energy to the microchamlel via a heated block. 

17. The method of claim 15, wherein the step of applying

a temperature gradient comprises cooling a portion of the

icrocharmel using the ambient temperature as a maximum 

emperature.

18. The method of claim 15, wherein the step of applying

a temperature gradient comprises supplying thennal energy 

o the microchanuel via a heated block and removing

hermal energy from the microchanuel via a cooled block. 

19. The method of claim 2, wherein the ionic buffer 

solution and analytes are contained within a capillary tube. 

20. The method of claim 19, wherein the step of establishing a temperature gradient comprises supplying thermal 

energy to the capillary tube via a heated block. 

21. The method of claim 19, wherein establishing a

emperature gradient comprises cooling a portion of the

capillary tube using ambient temperature as a maximum 

emperature.


22. The method of claim 19, wherein the step of establishing a temperature gradient comprises supplying thermal 

energy to the capillary tube via a heated block and removing 

hermal energy from the capillary tube via a cooled block. 

23. The method of claim 1, wherein said temperature 

dependent property is pH and said temperature gradient 

establishes a gradient in the pH of said ionic buffer solution, 

and whereby analytes are focused at a pH other than the 

isoelectric points of the respective analytes. 


24. The method of claim 1, wherein said temperature 

gradient establishes gradients in both the ionic strength and 

pH of the ionic buffer solution, and whereby analytes are 

focused at a pH other than the isoelectric points of the 

espective analytes. 

25. A fluidic device, comprising: 

a fluid conduit; 

an ionic bufier solution with a temperature dependent 

property selected from the group consisting of ionic 

strength atId pH disposed in said fluid conduit; 

an electric voltage source for providing an electric field 

within said fluid conduit, thereby causing one or more 

ionic analytes to have electrophoretic motion; 

t

r

t

m

t

t

t

t

t

t

r
US 7,029,561 B2 


13 

at least one heat source or heat sink, thermally coupled to 

said fluid conduit, for providing a temperature gradient 

having a significant component substantially aligned 

with the electrophoretic motion of one or more ionic 

analytes so as to form an electrophoretic velocity 

gradient within said fluid conduit; and 

a source of bulk fluid flow for providing flow of said ionic 

buffer solution in said fluid conduit, in a direction 

opposite to the electrophoretic motion of at least one of 

said one or more ionic analytes; 10 

whereby said at least one of said one or more ionic 

analytes will accumulate or be focused at at least one 

point along said temperature gradient, the pH at said at 

least one point being unequal to the isoelectric point of 

said at least one of said one or more analytes that are 15 

focused at said at least one point. 

26. The fluid device of claim 25, wherein said temperature 

dependent property is ionic strength and said temperature 

gradient establishes a gradient in the ionic strength of said 

ionic buffer solution. 20 

27. The fluidic device of claim 26, wherein said fluid 

conduit comprises a microchannel formed in a substrate, 

having a geometry with at least one spatial dimension on the 

order of micrometers, and where a temperature gradient is 

applied to said substrate. 25 

28. The fluidic device of claim 26, wherein said fluid 

conduit comprises a channel formed in a substrate and 

having a geometry with at least one spatial dimension on the 

order of at least one millimeter, and where a temperature 

gradient is applied to said substrate. 30 

29. The fluidic device of claim 26, wherein said fluid 

conduit comprises a chamlel formed in a substrate and 

having a geometry with at least one spatial dimension on the 

14 

order of at least one centimeter, and where a temperature 

gradient is applied to said substrate. 

30. The fluidic device of claim 26, wherein said at least 

one heat source comprises a power supply for applying an 

electrical current to said fluid conduit to thereby generate the 

temperature gradient in said ionic buffer solution by louIe 

heating. 

31. The fluidic device of claim 26, wherein said at least 

one heat source comprises a heated block for providing 

thennal energy to said fluid conduit. 

32. The fluidic device of claim 31. wherein said at least 

one heat sink further comprises a cooling block spaced from 

said heated block and thermally coupled to said fluid conduit 

for removing themlal energy from said fluid conduit. 

33. The fluidic device of claim 31, further comprising a 

thermally conductive adhesive disposed between said heated 

block and said fluid conduit. 

34. The fluidic device of claim 26, wherein said at least 

one heat sink comprises a cooling block for removing 

thermal energy from said fluid conduit. 

35. The fluidic device of claim 34, wherein said heat 

source comprises a power supply for applying an electrical 

current to said fluid conduit to thereby generate the temperature gradient in said fluid conduit. 

36. The fluidic device of claim 26, wherein said fluid 

conduit comprises a capillary tube. 

37. The fluid device ofclaim 25, wherein said temperature 

dependent property is pH and said temperature gradient 

establishes a gradient in the pH of said ionic bufier solution, 

and whereby analytes are focused at a pH other than the 

isoelectric points of the respective analytes. 

* * * * *