CA2284622A1

CA2284622A1 - Methods and systems for enhanced fluid transport

Info

Publication number: CA2284622A1
Application number: CA002284622A
Authority: CA
Inventors: Theo T. Nikiforov; Sang Jeong
Original assignee: Individual
Current assignee: Caliper Life Sciences Inc
Priority date: 1997-04-04
Filing date: 1998-03-30
Publication date: 1998-10-15
Also published as: ATE283571T1; US6471841B1; AU6788798A; ES2234107T3; AU739133B2; DE69827781T2; EP0972333B1; WO1998045929A1; EP0972333A1; EP0972333A4; US5964995A; DE69827781D1; US6129826A

Abstract

The present invention generally provides methods for enhancing transport and direction of materials in fluidic systems, which systems utilize electroosmotic (E/O) flow systems, to affect that transport and direction. The methods generally comprise providing an effective concentration of at least one zwitterionic compound in the fluid containing the material that is to be transported or directed.

Description

WO 98/45929 PCT/US9$/06256 VfETHODS .~~ID SYSTEMS FOR E~IHAUICED FLUID TRAU1SPORT
BACi~G:.OUND OF THE INVENTION
There has beer.a growing interest in the development and manufacturing of microsca:.e fluid systems for the acauisition of che_Tnicai and biochemical :.nfornation, in both preparative and analytical capacities. .'-.daptation of technologies from the electronics industry, suc:: as photolithography, wet chemical etching and the like:, has helped to fuel this growing interest.
IO One of the tirs~ areas in which microscaie fluid systems have been used for c:hemic~:i or biochemical analysis was in the area of capillary ei.ectrcphoresis (CE). CE systems generally employ fused silica capil=_aries, or more recently, etched channels in planar silica substrates, filled with an appropriate separation 1~ matrix or medium. f~ samp=_e fluid that is to be analyzed is injected at one end of t'~e: capillary or chanr_el. Application oz a voltage across the c:apill~:ry then permits the electrophoretic migration of the species -,~ithin the sample. Differential electrophoretic mobilitiea of the constituent elements of a sample 20 fluid, e.g., due to their differential net charge or size, permits their separation, icientif_cation and analysis. In order to optimize the separation a:.pect of the CE applications, researchers have sought to maximize t':e electrophoretic :nobility of charged species relative to each other and relative to the flow of the luid through the capilla~_-y resulting from, a.g., electroosmosis.
See, e.g., U.S. Patent No. 5,015,350, to Wiktorowicz, and U.S.
Patent No. 5,192,401 to P~stersen et al.
In comparison to these CE aplications, the technologies of the electronics :_ndust:~r have also been focused on the 30 production of small scale fluidic systems for the transportation of small volumes of fluids over relatively small areas, to per~orn one or more preparative c:= analytical manipulations on that =laid.
These non-CE 'luidic: syst_:ns differ from the C.E systems in that their goal is not tile elegy=trophoretic separation of const-_tuents of a sample or _luid, but is instead directed to the bulk transport of fluids ar_d the materials contained in those fluids.
Typically, Chess non-C. rluidic systems have relied upon mechanical fluid direction and transport systems, e.g., miniature pumps and valves, to affect material transport from one location to another. See, e.g., Published PCT application No. 97/023:7.
Such mechanical systems, owever, can be extremely difficult and expensive to produce, and still fail to provide accurate fluisic control over volumes that are substantially below the microl_t'r r ange .
Elect=oosmotic (E/O) flow systems have been descr_bed which provide a substantial ;mprovement over these mechanic~~
systems, see, e.g., Published PCT Application No. w0 96/0457 to Ramsey et al. ~_'ypicall=r, such systems function by applying a voltage across a fluid f_lled channel, the surface or walls of which have cha=ged or ionizeable functional groups associate therewith, to produce electroosmotic flow of that fluid in t.e direction of the current. Despite the substantial improvements offered by these electroosmotic fluid direction systems, there ?0 remains ample room for improvement in the application of these technologies. The present invention meets these and other needs.
~Rv F mug T~1T ON
SLJM~? _ . _ 0_ -r The present i=wention generally provides methods, systems and devices which provide for enhanced transportation~_ and direction of materials using electroosmotic flow of a fluid containing those materials. nor example, in a first aspect, the present invention provides methods of enhanci.~.g material direction and transport by elect=cosmot-c flow of a fluid containing t.at material, which method comprises providing an effective concentration of at least one zwitterionic compound in the _=uid containing the mater_al.
In a relates aspect, the present ~r_vention also cr~vides methods of reduci_~_g electrcDheretic separation of diffcre nt~.~__y charged species in a mic:roscaie fluid column, whera that fluid column has a voltage app>iied across it, whicmethod comprises providing an effective concentration of at yeast er_e zwitterionic compound in the fluid.
The pre~~ent invention also provides mic=ofluidic systems which incorporate these enhanced fluid direction a.:d transport methods, i.e., provide for such enhanced fluid transport and direction within a microscale fluid channel struc~vre. In particular, these microfluidic systems typically i=elude at least three ports disposed at t:~e termini of at least tNo intersecting fluid channels capable of supporting elect=oosmoti~ flow.
'I'vpically, at least one of the intersecting channe_s has at least one cross-sectional dimension of from about 0.1 um to about 500 um. Each of the ports may include an electrode placed in 1~ elect=ical contact with it, and the syste_,n also includes a fluid disposed in the channels, whereby the fluid is in electrical contact with those electrodes, and wherein the fluid comprises an effective concentration of a zwitterionic compound.
~0 _EiRIEF I)ESCR_TPTION OF TuE "'TGURE~
Figure 1 is a schematic illustration of the effects of electrophoretic mobility of charged species on the migration of those species in a coherent electroosmotic fluid flow. Figure 1~
illustrates an optimal scenario where differentially charged '_'f chemical species contained in discrete fluid volumes have apparent mobilities that ar~= substantially the same as the electroosmotic flow rate for the fluid. Figure 1B illustrates the situation wherein the appare:at mobility of positively charged species is greater than the rate of electroosmotic flow and to apparent 30 mobility of negatively charged species is less than or opposite to the rate of electr«osmotic flow, resulting in the electrophoretic biasing of the charged species within the discrete fluid volumes.
Figure 1C illustrates the situation where the apparent mobilities of carged species are substantially different from the rate e.

electroosmotic flow of the rluid, such that the charged species in the two discrete fluid volumes overlap.
Figure 2 is a graph showing the effect of the addition of sulfobetaine on electroos:,iotic flow ar_d apparent mobility of charged species, under conditions of electroosmotic flow.
Figure 3 illustrates a microfluidic device used to perforn enzyme inhibitor assays.
Figure ~ illustrates a graphical comparison of enzyme inhibition assays i:~ the presence and absence of a zwitterionic compound, NDSB.
DEVILED DB'~C~'DTTON OF 'T'w= r~l~~7mTpN
I . Ger_er a1 The present invent=on generally provides methods and 1~ systems for the enhanced transportation a_nd direction of materials within fluidic systems, which utilizes the electroosmotic flow of fluids containing those materials . By ~~e_~ar_ced transportation and direction" is generally meant the electroosmotic flow and direction of fluids withi_~_ ~luidic systems, which shows: (1) a reduction in the electrophoretic mobility of a charged species relative to the electroosmotic flow of the _luid containing that charged species; and/or (2) az increase in the overall electroosmotic flow of that rluid, relative tc such systems not incorporating the present invention, as described herein.
'-~ A. Reduction. o' Electroohoretic r?obi 1 itv or Charcred ~z~ecies As noted previousi=r, in capillarty electrophoresis applications, the general goal is to maximize the separation between different species cor_tained in a sample of interest, in order to separately analyze those species, identify their presence within the sample, or the lice. This is accomplished by maximizing t he differences ___ the a l ectrophoretic mobilities of these species, which differences may result ..nom differences in their size and/or net c?:arae.

In the E/O fluid direction systems described herein, however, the goal~~ are somewhat different from those of CE
systems. =n particular, the general object cf these E/0 fluid directior_ s~rstems is the transport and/or direction of material of interest. contained in a volume of fluid or.multipie discrete volumes of fluid, from one location in the system to another, using cor_~rolled E;/0 flow. Because these rluids are generally to be subjected to further maripu_ation or combination with other fluids, it is gene:rail~r desirable to affect the transportation of these fluids withcut substantially altering their make-up, i.e,, electrophoretically sepa.rati_-g or biasing differentially charc_ed or sized materials conta.ied w=thin those fluids.
Similar 3.y, whE:re tL-:ese systems are beir_g used to serially transport small volumes of fluids or multiple discrete l~ volumes of different fluids along the same channels, it is generally desirable to transport these fluid volumes as coherently as possible, i.e., minim=zir_g smearing of materials or diffusion of fluids. In particular, because these systems are preferably utilized in microfl~~idic applications, the improved coherency of a '0 particul ar fluid volume svith_:, the E/O flow system permits the transport of larger numi~~ers of different fluid volumes per unit time. Specificall~l, mai::~tai =ing higher fluid volume coherency allows separate vo:Lumes to be transported closer together through the channels of they sys to=_m, :vi shout resulting in excessive f intermixing of there voiu.mes. :urther, maintenance of maximum fluid volume coherency during the transport and direction of the fluids permits morE: prey:LSe control of volumetric delivery of materials within t;'iese systems.
Despite the di.~feri= g goals of the C~ systems and the 30 ~/0 flow s~rstems u~~ed i= the present inventio:_, in each case, the application of an e~lectr~.cal __'ld across a fluid of interest has the same basic result. "peci=_cally, where the fluid of interest comprises c::arged specie; , cr ;~ made up of a plurality of differentially charged c.emica_ species, appl'_cation of a voltage across that fluid, e.g., to obtain E/0 flow, Taill result in those charged species electrophoresing within tile ~?uid, and the differentially charged species electrophoresi~g at different rates. As such, in a channel having a negative surface potential, negatively charged species will have an electrophoretic mobility opposite to the direction of E/0 flow, whereas positively charged species will have an electrophoretic mobil=ty in the same direction of E/0 flow. The greater the number of charges a particular species has, the greater its electrophoretic mobi'_=tv in the same or opposite direction of E/0 flow. In systems employing electroosmotic =luid direction, this results in a r_et separation of differentially charged species that are contained within the fluid that is being transported.
Where one is transporting a particular volume of a given I5 sample fluid, this separation can result in an electrophoretic biasing of the sample, where the positively charged species have a greater apparent mobility, than negatively charged species.
"Apparent mobility" as used herein, generally refers to the overall mobility of a given species within the =luidic system. In the systems of the present =nvention, apparent mobility is typically defined as the rate of E/O mobil_ty plus the electrophoretic mobility. Where electrophoretic mobility is opposite to the direction of E/0 flow, i.e., negative, this leads to an apparent mobility that is less than t=a E/O mobility.
15 In the case of species having high eiectrophoretic mobility, e.g., highly charged species, the e~~ect can be magnified to the point that the apparent mobility of such sr~ecies is substantially differer_t from the E/0 mobil_ty of the fluid contair_ing them. For example, species possess=ng multiple negative charges may have an electrophoretic :nobility substantially opposite the d'_rection of E/0 mobility, result_ng in a substantial reduction in the apparent mobil_ty of that species.
Where that reduction is sufficiently large, ~t can result in that species being effectively "left behind" by the particular volume of fluid t!~,at is being transported.
Conversely, a. species bearin mult~
g ~ple positive charges may have an appar~=_nt mobility that is far greater than that of the fluid being transported and other species contained therein, such that the species :is transported well ahead of the fluid volume.
This problem is not as significant where one is transporting large volumes of fluid from one location to another.
Specifically, one can reduce the effects of the electrophoretic separation of a f7.uid :.fir coll ecting 1 arger volumes, thereby reducing the contributrion that biased por=~.ons of the f?uid have on t he overall f 1 uid de 1. i vered .
riowever, the problem is substantially magnified when one wishes to transport a relatively small volume, or multiple small volumes of the same or c.ifferent fluids, without separating the materials contained in the individual fluid volumes or intermixing the materials contained in separate volumes. Specifically, in transporting a one or a series of discrete volumes of a particular fluid or fluids, e.g., samples, test compounds, various elements 30 of a screening system, species that have apparent mobilities that are substantially ~=ffer,=_nt from the E/0 mobility of the particular =luid volume will travel ahead of, and behind the fluid volume, effectivel~r smea:=. ing the materials that are sought to be delivered. ~s desc::ibed above, this is a significant disadvantage '-5 where relatively precise fluid control is desired, or where smaller effective ~rolume_~ are used. For example, where one is screening fer compoundss which affect a particular reaction mix, e.g., a biochemical. syste_.n, it is generally desirable to be abnle to mix the elements necessary for that screen,- e.g., enzyme, 30 substrate and test inhibitor, and allow those elements to incubate together while transporting them to the ultimate detection area.
Where those elements separate based upon their differential electrophoretic mobilites, this can have substar_tial adverse effecis or. the overall rT~ .
e_..ycacy of the screen~:.g system.

More importantly, where a species ir_ a first volume being transported has an apparent mobility that is substanti~ily less than the E/0 mobility of the fluid, while a species in a second or following volume has an apparent mobility that is substantially greater than the E/0 mobility of the fluid that is beivg delivered, those two species can overlap within the flow system.
The above described problems are schematically illustrated in Figure 1. Figure lA shows an optimal situation where discrete volumes or regions of fluids in a channel (fluids ~~ and Xy, shown underlined) contain differentially charged species, e.g., X+ ar_d Y-, and A+ and B-. In this optimal situation, these differentially charged chemical species have an apparent mobility that is not substantially different from the E/O
mobility of the fluid containing those species. As a result, the various species are maintained substantiall~r within their separate fluid regions. Figure 1B illustrates the smearing effect which results when charged species, as a result of their greater electrophoretic mobilities, begin to migrate outside of their respective fluid volumes or regions. This results in a smearing of the materials that are being transported and substantialv reduces the precision with which these materials can be transported. Finally, Figure 1C illustrates the situation where the apparent mobility of the charged species is so substantially '-5 different from the E/O mobility of the fluid r=gions, that it results in the overlapping and intermixing of differentially charged species from different fluid regions. The intermi:cing of separate fluid volumes creates substantial problems where the fluid system is beir_g used i.~. the serial transport of multiple 30 different fluids, e.g., as described in U.S. ?atent Application No~ 08/761,575, filed Dece.Tnber 6, 1996, and =:corporated herei= by reference in its entirety for all purposes.
Methods have been developed to prevent and/or correc~
for the excessive electrophor'tic mobility of charged specis, when those speci:_s are being transported in E/0 fluid direct'_on systems, by incoz-porat:ing fluid barriers aroun d the fluid being transported, in ~~rhich t_he eiectrophoreti c mobility of these charged species i.s substantially reduced, see, e.g., commonlv assigned U.S. Pat.ent Application Serial No. 08/760,446, filed December 6, 1996, and i.ncor~orated herein by reference in its entirety for all purpo_;es.
Genera~.ly, the er~nanced E/0 material transport and direction produced by the present invention is carried out by l0 providing within the fluid component of t~:e system, a compound or compounds that are capable of reducing the effects such an E/O
system has on charged species contained within the fluid. For example, incorporation of these compounds within the fluid component of the ~3/O flow system typically results in a reduction 1~ in the electrophoretic mobility of charged species, and thus, reduces the dlff2?:ent3.a.1 electrophoretic mobility and apparent mobility of diffe::entia:Lly charged species .
In prefs=_rred aspects, zwitterionic compounds or combinations thereof, a~_-e used to reduce the electrophoretic ?0 :nobility of mater;.als t~:at are contained within the fluids th at are sought to be c.ransported using these ~/0 fluid direction systems, thereby a.chievi.ng or substantially achieving the optimal situation shown in Figure ?a.
Without being bound to a particular theory of operation, '-5 it is believed that such. zwitterionic compounds interact with the charged species in a layer-like complex. "'he ~~complex" has the same net charge as the charged species, but that charge is spread over a much larger structure effecthrely reducing the charge: size ratio, and r2dL1C1n(~ the electrophoretic mobility of the complex.
30 Secause z-aitterior_:~ are Bipolar molecules, they can be effectively employed with res~e~ct to pos_tively or negatively charged species.
Y~lhile ot:zer methods can be used to effectively reduce the charge: size ratio of compounds in an E/O fluid direction System, these methods hare numerous associated problems. For example, raising or lowering pH of the fluid containing the species can effectively reduce the level of charge of a chemical species by protonatirg or deprotonating functional groups present thereir_. 6~Thile effective in reducing net charge of a given species, this method can 'nave substantial adverse effects.
Speci~ically, where the fluidic system is being utilized in the analysis of biological systems, e.g., enzymatic reactions, receptor/ligand interactions, or in transporting other materials sensitive to extremes or pH, the substantial variation of pH, e.g., from neutral or physiological conditions, can place the system well outside the optimal pH for subsecruent manipulation or analysis. In some cases, t_~e optimal pH for reducing the net charge of a particular species may denature or otherwise degrade active components of the materials that are being transported.
The incorporation of zwitterionic compounds as described herein, on the other hand, is readily compatible with syste.Tns to be used for the transport o= pH sensitive materials, e.g., systems used in analysis of biological systems. In particular, different zwitterionic compounds, i.e., having different pI, may be selected ?0 dependi:=g upon the pH sensitivity of the material being transporred. According~y, as can be readily appreciated from the foregoing, the present inveTtion is particularly useful in E/0 fluid direction systems when' the materials to be transported include biological material, such as enzymes, substrates, l;gands, '-S receptors, or other elements of biological or biochemical systems, e.g., as those systems are defined in U.S. Patent Application No.
08/761,575, previously incorporated herein by reference for all purposes.
Another method that can be used to affect the 30 charge:size ratio of a charted molecule of interest in an E/O
fluid direction system involves interacting that charged molecule of interest with another mo_ecule or species such that the two molecules form a complex having a different charge: size ratio.
:Merely by way oT example, =_~orescein is a molecule that carr_es two negative charges above neutral pH. The elect~ophoretic mobility of this molecu.ie can be readily altered by adding an antibody, such as anti-=iuorescein to the solution. The resulting complex will have a substantially reduced electrophoretic mob~litv_ over that of fluorescein alone. again, while this method is effective, it too carries a number of disadvantages. First, because one must identify a compound that associates with the charged molecuie~ ~~f interest, a specifical I=r associating compound must be identified for each charged molecular species in the f luid , and f or eac~ h di f f er en t f luid us ed ir_ the sys tem . Fur t her , as is t~ne case wi?_h the fluorescein/anti-= 1 uorescein comtzlex described above, ~rcorp~~ration of an active molecule into a larger complex can have an advc=_rse effect on the on the desired acti-rity or function of that molE~cule, i.e., substantially reduced 1~ fluorescence.
The metlZOds and syste_ns of the present ~ nvention, or_ the other hard do not have these associated problems. For example, the f~,:nction of z~~iitter=_oric compounds ir. reducing electrophoretic mobility of charged species is generally applicable, i.e., does ?~ not require a sped=is interaction between t.e charged species and the zwitterion. ~'urthen, the nature of thi s inter actior_ resu_ts in littl a or no eTfect on the properties o~ the ch arged molec~.:le of interest.
B. Increase' =n E/O ~ob~~li~y '-5 T_n addit:ion to the advantages of reducing the electrophoretic mobility of charged species within fluids that are being transported using E/O fluid directions systems, incorporation of zwitterionic compounds in ma.~y systems can a_so have the effect of increasing the E/0 mobility in the fluid 30 direction system, thereby further optimizing the apparent mo:oi'_itv of the material that is beir_c transported.
In particular, incorporation of zwitterionic compoun ds within r'_~,:ics being transported in E/0 fluid direction systems has been shown ~o incre_ase E/0 :nobility of those =1 uids . This e==ect is particularly apparent where those fluids include a protein component or other larger charged molecular species.
I_T. Comnour_ds Useful in Practicina the Invention A wide variety of zwitterionic and relates chemical compounds may be employed according to the present ~:vention. For example, such compounds include, e.g., betaine, sulfobetaine, taurine, amir_omethane sulfonic acid, zwitterionic am=no acids, such as glycine, alanine, f3-alanir_e, etc. , and other zwitterionic compounds such as f~EPES, MES, CAPS, tricine and the 1~ke. In part~c~larly preferred aspects, non-detergent, low molecular we=ght sulfobetaines are used in the methods of the preser_t invention, such as dimetiz~rlethylaminopropane sulfonic acid, dimet'~ylber_zylaminopropane sulfonic acid, and 3-(N-1~ pyridinium)propane sulfonic acid.
Although generally described in terms of s_ngle species of zwitterionic compounds, it will be readily appreciated that the present invention also comprehends the use of combir_atior_s of the above described compounds. Such combinations can be readily tailored to optimize the effects seen on the overall fluidic system, as well as for their computability with the various compor_ents of the system, e.g., buffers, enzymes, substrates, receptors, ligands, test compounds, and the like.
Generally, the concentration of zwitterionic compounds 2~ wit:~i=. the fluids contained in the system, may be varied dope.~_ding upon. the effect desired, where lower the concentrations yield less of an effect in reducing electrophoretic mobilities of materials contained within the fluid. Further, these effects may also be varied depending upon the nature of the charged spec=es contained 30 withi=~ the material of interest. Therefore, as uses herein, ~he te~-zn "effective concentratior_~~ refers to a concentration of zwitt=erior_ic compounds that is sufficient to achieve a desired effect, and particularly, achieve some reduction i the elect_ophorotic mobilit~r of a charged species of interest.

Further, by "concentration" of zwitterionic compounds in the fluid" is meant t:he amount of such compounds added per unit volume, ~egardle;~s oz any subseauent conversion of such compounds within the fluid system. 'T'~rpically, however, effective concentrations oi: zwitt:erionic compounds will preferably be greater than about 5 mbi, typically greater Khan about 10 mM, and often greater than about 50 mM. Although zwitterionic compounds may generally be present at levels approach.:g their solubi'_ ty limits in practicing t~.e present invention r r pre_e~red concentrations of the zwitterionic compour_ds in the fluid than is sought to be transported within the system will range between about 1 mM and 2M and more preferably between about 5mM and 2 M, IIT . ~DD1 ~ cation to Mic_rof i t:id.~ c wste~ns 1~ As noted previously, the prese_~_t i=lvention finds particular utilit~r in fluidic systems that employ E/O fluid direction systems, and more particularly, microscaie fluidic systems. By "E/0 fluid direction systems" is generally mear:t fluidic systems teat area made up of fluid channels or passages, ?0 chambers, Sorts oz- the ~.i:te, wherein the movement of ~-din fluid w._:
the systems, i.e., through the chanr_els, or from one channel ~o another channel, or from one chamber to ar_other chamber, is selectively directed through the controlled electroosmotic glow of that fluid. Examples of sac:: controlled ?/0 flow systems ar=_ ?~ described in, e.g., Published PCT ~pplicaton No. WO 96/04547, and commonly owned U.S. Patent Application Nos. 08/761,575 and 08/760,446, each of whic:~ was previousl~r incorporated here~a Lv reference. -In preferred aspects, such fluid direction systems 30 direct a fluid of inter eat through intersecting channel struct~.:res by applyir_g a voltage gradien~ along the cesired path of fluic flow. Voltages are. t_,rpicaily simultaneously applied alone intersecting fluid paths, in order to propagate a containing c.
directing fluid flow, i .:a. , to contai ; ~~,- _~ ~, . -n or c~r...._ ~::e __u~d c~

interest along the desired path. For example, where the fluid of interest is being flowed along a =first channel that is intersected by second channel, the flow of the fluid of interest is maintained within the first channel, i.e., prevented from diffusing into the intersecting channel, by simultar_eously flowing fluid into the first channel from each side of t'_:e intersecting channel. This is generally done by simuitaneousl~r applying a voltage from the originating end to the terminate.~_g end of t'_:e first channel, and to each end of the intersecting channel, whereby appropriate ~/0 IO Flow is obtained. ~s can be appreciated, this results in a f_uid flow pattern in the first channel that appears "pinched.° In another example, a fluid of interest :nay be directed from a first arm of a first channel into a first arm of an intersecting channel, by applying a voltage across the desired fluid flow path 1~ to generate fluid flow i_~_ that direction. In order to control fluid flow at the intersection, a containing =luid flow is generated along the entire length of the intersecting channel creating what is termed a "gated" flow. The fluid of interes:. can then be metered out or dispensed i.n a controlled fashion, into the '_'0 remaining a~-in of the first channel by activel=r modulating the voltage to allow the fluid to flow into that arm, while preven~ing diffusion. ~'ffectively, this results in a val wing system wit: out the necessity of mechanical elements. =finally, by modulating the rate of flow of the fluid or interest through an intersection as ?5 compared to the flow of diluents 'lowing in from the intersect'_ng channels, these systems can be used as diluters.
By "microscale fluidic systems" is typically meant =iuid systems that comprise reservoirs, conduits or channels, and/or chambers, wherein at least one cross sectional dimension, e.g.
30 depth, width or diameter, of a particular Fluid channel and/or' chamber is in the range of from about 1 urn'to about S00 um, inclusive. Such microscale _luidic systems range from simr_ole capillary systems, e.g., that employ a single fused silica capillary =or delivering a particular Lluid c. fluids from a I -.E

reservoir at one end of the capillary to t::e other end of the capillary, for analysis, combination with other reagents, and the like, to more complex i ntegrated multicharr_el microfluidic de~rices fabricated in solid substrates, such as those described in U.S.
Patent application Serial No. 08/761,575, previously incorporated herein by referen~~e in its entirety for all purposes. In preferred aspects, the microscale fluidic system wilt employ at least one channel, and :pore preferably at least two intersecting channels which hare at 'east one cross sectional dimension in the range from 1 ~,un to abouv 500 um, and more preferably bet-aeen about 1 um and 10 0 ~.un .
The com:binatior_ of these microscale dimensions with the relatively precise flu'_d control, described above, permits the controlled, repeatable <irection or dispensing of extremely small volumes of fluid, whic~ volumes are dictated by the volumes o~ the channels and/or intersections, e.g., a sample plug at an intersection, or by the timing of fluid flow, e.g., the amount of time or length of a fl~.:~.d plug injected ir_to a channel using gated flow.
~0 Typical:Ly, thEa mic=ofluidic systems empl oyed in practicing the present ~.nvention will comprise a solid substrate that has the channels ar.~d/or chambers of th a microfiuidic system disposed within it. Substrates may be prepared from a number of different materials. For example, technicues employed in the fabrication of small sca.ie f~uidic devices are often derived from the electronics industr;u. As a result, substrate materials are often selected for compatability with these manufacturing tecr~:iques, such as silica, silicon, gall=um arsenide and the like. Typically, however, semiconducting materials are not preferred for practicing the present invention, as they are not compatible with the application of electric f~elds through fluids, without some modification, e.g., application of an insulating layer . Accordingl:y, i n one preferred aspec t , s i 1 ica subs tr ates are preferred in p:ract=:ing the present ~ nve_~_tion.

Other substrate materials mav_ also be employed in the microfluidic systems of to invention, and may generally be selected for their compatibility with the conditions to which they will be exposed, both in manufacturing, e.g., compatibility with S known manufacturing techniques, and operation, e.g., compatibility with full range of operati:.g conditions, including wide ranges of salt, pH, compositions, and application of electric fields.
Examples of such substrates include polymeric materials, with the provision that such materials, either on their own, or through modification of the surfaces that contact the fluids of the system are capable of propagate~g E/0 flow.
Typically, the substrate will have a first surface, and will be generally planar in shape. The intersecting channels are typically fabricated into the surface of the substrate as grooves.
1~ as noted previously, the c'_hannels may be fabricated into the surface of the substrate using, e.g., photolithography, wet che.rnical etching, and other known microfabrication techr_iques.
Generally, a cover layer is overlaid on the surface of the substrate to seal the grooves, forning fluid channels or passages.
0 The devices generally include a number of ports or reservoirs fabricated therei~, which ports are in electrical contact, and typically in fluid communication, with the intersecting channels. These ports generally provide a point at which electrodes can be placed in contact with the fluids, for ~5 directing fluid flow. These ports also often provide a reservoir of fluids that ire used in the device or system. As such, the different ports are typically placed in contact with the fluid channels on different sides of a given intersection of two channels. For ease of fabrication, such ports are typically 30 placed in electrical contact with each of the free termini of the various chanr_els fabricated into the cevice. By ~~free term=ni" or "free terminus" is meant a nonintersected terminus of a chanr_el.
For ease of discussion, the microfluidic devices and systems are Generally descr_bed in terms of two i:tersectina channels. Howeve:_, it will be readily appreciated that such devices and systems may readily incorporate more complex channel structures of thrEae, four, five, ten, twenty and more intersecting channels. Further, such devices and systems also include parallel channel structures where more than one main channel may be intersected by large numbers of cross channels.
As described above, the present invention generally relates to methods of enhancing electroosmotic flow, and particularly, app7_ication of these methods to microfluidic systems which utilize such E/0 i_low in the transport and direction of fluids within the~~e systems. This is in contrast to capillary electrophoresis s~~stems (CE) which seek to minimize E/O mobility of fluids, while maximizing differential electrophoretic mobility of species contained in these fluids. Often this is done by incorporating a separation matrix within the channels of the CE
systems, which furthers these goals. Thus, the presently described systems are generally described in terms of channels which permit or are capable of free electroosmotic flow. By this is meant that the channels in which E/O flow is desired will generally have a sufficient surface potential for propagating E/O
flow or mobility of fluids and materials in those channels. At the same time these char.~nels are devoid of obstructions which might impede that flow, and particularly such channels will be free of any separation media or matrices.
The prey>ent invention is further illustrated with reference to the following non-limiting examples.
FXAMPLE~
The efficacy of incorporating zwitterionic compounds for reducing electrophoretic mobility of charged species in E/O flow systems was demonstrated in a fused silica capillary, having 57 cm total length, 50 c:m effective length and internal diameter of 75 ~.un. All samples were run in 50 mM HEPES buffer at pH 7.5. All of the running buffers were prepared fresh from concentrated stock solution. For each run, the samples were pressure injected into the capillary for 20 seconds, separated at 30 kV, and detected at 254 nm. Following each run, the capillary was rinsed with 1N NaOH
for 2 minutes followed by a 5 minute rinse with replacement buffer.
The level of electroosmotic flow within the capillary was determined by incorporation of mesityl oxide (4 ul in 4 ml Hz0), a neutral detectable marker, while effects on electrophoretic mobility were determined by incorporation of 5.0 mM dFMUp (6,g_ difluoro-4-methylumbelliferyl phosphate) in water, a detectable compound having two negative charges at neutral pH.
Example 1: Use of NDSB-195 The first experiment tested the effect of the zwitterionic compound 3-(N-ethyl-N,N-dimethylammonium) propanesulfonate) (NDSB-195) on electrophoretic mobility of charged species (dFMUP) within a buffer filled capillary, as well as on overall electroosmotic flow of that buffer within the capillary. The experiment was duplicated in the presence and absence of a protein component (0.1 mg/ml BSA).
Three different concentrations of NDSB-195 were tested:
0.1 M; 0.5 M; or 1.0 M final concentration, and compared to a negative control (no NDSB-195). For each run, the retention time of mesityl oxide and dFMUP was determined, and used to calculate the E/0 mobility for the run (uE0), electrophoretic mobility (uEP) of the dFMUP, and the apparent mobility (uApp) of the dFMUP
(tiAPP=uE0+uEP). The results are shown in Table I, below, as averages of triplicate runs:

Table I
[NDSB] uE0 uE0 1~PP I~PP uEP ~1EP

M X 10 ' X 10' X 10 X 10 X 10 X 10 (+BSA) (- B.~.~A)(+BSA) (-BSA) (+BSA) (-BSA) 0 2.94 4.7'7 0.20 2.02 -2.74 -2.75 0.10 4.31 5.4:? 1.54 2.87 -2.78 -2.55 0.50 5.74 5.59 3.49 3.34 -2.24 -2.24 1.00 5.68 5.20 3.60 3.28 -2.08 -1.92 Figure 2 shows a plot of E/O flow, electrophoretic mobility and apparE~nt mobility of dFMUP, as a function of increasing concentration of NDSB-195, both in the presence and absence of BSA. The standard deviation is also shown for each point plotted. As is apparent from these data, inclusion of NDSB-195 substantially reduces the net electrophoretic mobility of dFMUP, both in the absence and presence of a protein component (BSA). In addition to reducing this electrophoretic mobility, the incorporation of 1VI)SB al:>o increases the E/O flow rate of the system. The net result :s that the apparent mobility of the charged species is brought closer to the E/O flow rate of the system.
Example 2: Use of i~~- an''e A similar- experiment was performed utilizing an amino acid, Q-alanine, as the 2;witterionic component. In particular, i3-alanine was incorporated in the same system as described above, at two different concentrations, 0.50 M and 1.0 M, and compared to a negative control (contair..ing no !~-alanine), and in the presence and absence of a protein component. pH was not adjusted following addition of i3-alanine. T'he results of this experiment are shown in Table II, below:

Table II
Buffer Analyte R.T. ixEO pppp_ ~p X 10~' X 10 ' X 10' 50 mM HEPES, Mes. Ox. 3.41 4.64 4.64 pH 7.5 8.61 1.84 -2.80 50 mM HEPES, Mes. Ox. 3.90 4.06 4.06 pH 7.5/BSA

( 0 .1 mg/ml ) dFl~tCTP 14. 1. 07 -2 . 99 50 mM HEPES, Mes. Ox. 3.00 5.2B 5.28 pH 7.5/BSA

( 0 .1 mg/ml ) / dF'~1UP 5 . 2 . 91 -2 . 37 500 mM ala.

50 mM HEPES, Mes. Ox. 3.00 5.28 5.28 pH 7.5/500 mM

ala. dFMtlP 5.51 2.87 -2.40 50 mM HEPES, Mes. Ox. 2.94 5.39 5.39 pH 7.5/BSA

(0.1 mg/ml)/ dFMUP 4.94 3.21 -2.18 500 mM ala.

50 mM HEPES, Mes. Ox. 2.96 5.35 5.35 pH 7.5/500 mM

alanine dFTILTP 4.99 3.I7 -2.18 R.T.= retention time (mins) Again, incorporation of !3-alanine resultsin a decrease in the electrophoretic mobility the dFMUP, and net increase of a in its apparent mobility.

Example 3: Concurrent Application to DifferentiallyCharged Species.

Each of the above examples illustrates that the incorporation of zwitterionic compounds results in a reduction of the electrophoretic mobility of negatively charged species in the system (dFMUP). 'rhe following experiment illustrates the same efficacy in sample=_s containing both positively and negatively charged chemical :~pecie:a .
This experiment tested the effect of 1 M NDSB on the electrophoretic mobilit~t of a positively charged species (benzylamine) and a negatively charged species (benzoic acid) in the same capi11ar5r syste=m described above. This experiment utilized two different buffer systems: 200 mM borate at pH 8.7;
and 200 mM HEPES at pH ',~Ø These experiments also incorporated dimethylforrnamide (DMF) as a neutral marker compound, for ascertaining E/O mobilit:y.
Table 3, below, illustrates the effect of incorporation of the zwitterioni.c compound NDSB on the electrophoretic mobility and apparent mobility of: both positively and negatively charged species.
Table III
Buffer Analyte R.T. ~p app X 10'' X IO'' X 10-' 200 mM Borate ~' 3.05 5.19 5.19 --pH 8.7 Henzylaminf~ 2.04 -- 7.76 2.57 Beizzoic Acid 7.13 -- 2.22 -2,97 200 mM Borate Lid 3.26 4.86 4.86 --pH 8.9, 1M NDSB
Benzylamine 2.47 -- 6.41 1.55 Benzoic Acid 5.43 -- 2.92 -1.94 200 mM HEPES Ltd' 2.97 5.33 5.33 --pH 7.0 Bemzylamine~ 1.93 -- 8.20 2.87 Ber.,zoic Acid 5.25 -- 3,02 -2.32 200 mM HEPES ~' 3.56 4.45 4.45 --pH 7.0, 1M NDSH
Henzylamine 2.40 -- 6.60 2.15 Benzoic Acid 5.84 -- 2.71 -1.74 From these data, it is clear that incorporation of the zwitterionic compound NDSB in either buffer system reduced the electrophoretic mobility of both the positively charged species, benzylamine, and the negatively charged species, benzoic acid.
Further, although in this system, NDSB resulted in a decrease in the E/0 flow rate, there was nonetheless, a reduction in the difference between the E/0 mobility and the apparent mobility for both of the differentially charged species, e.g., the apparent mobility of the positively charged species was reduced while the apparent mobility of the negatively charged species increased.
Example 4: Enzyme Inhibition in Presence and Absence of NDSB in Microfluidic System.
An enzyme inhibition assay was performed using a microfluidic device having a well/channel structure as shown in Figure 3. Standard semiconductor photolithographic techniques were used to etch channels 70 um wide and 10 um deep, in a 525 um thick soda lime glass substrate, and a second 2 mm thick layer of glass having 3 mm diameter holes drilled through it, was thermally bonded to the first, providing the various wells.
All reagents were diluted in the same buffer solution which also served as the running buffer: 25 mM HEPES, pH 7.9, for the control; and 25 mM HEPES, 1 M NDSB-195 (non-detergent sulfobetaine, MW 195)(available from Calbiochem-Novabiochem, LaJolla, CA), pH 7.9, for the test run. The assay solutions were prepared from stock solutions of 5000 U/mg leukocyte antigen related phosphatase (LAR) (enzyme)(New England Biolabs, Beverly, MA), 10 mM dFMUP in water, a fluorogenic substrate for LAR
(substrate)(available from Molecular Probes, Eugene, OR), and 1.4 mM of a known competitive inhibitor of LAR (inhibitor).
Detection of fluorescence was carried out using a Nikon inverted Microscope Diaphot 200, with a Nikon P101 photometer controller, for epifluorescent detection. An Opti-Quip 1200-1500 50W tungsten/halogen lamp coupled through a I0X microscope objective provided the light source. Excitation and emission wavelengths were selected with a DAPI filter cube (Chroma, Brattleboro VT) fatted with a DM400 dichroic mirror, 340-380 nm excitation filter and 435-485 nm barrier filter. Reagent well currents and volt<~ges on the chip were controlled using a Caliper 3180 Chip Control:Ler (Palo Alto, CA). The currents and voltages ranged +/- 10 ~zA ~~nd 0-2000 V. Data was collected on a Macintosh Power PC 7200/120.
The channels of the device were filled with running buffer by placing the buffer in a buffer well and allowing capillary action t=o distribute the buffer throughout the channels.
125 nM LAR enzyme was placed in the enzyme well, 50 uM dFMUP was placed in the substrate well and 200 ~.iM of a known competitive inhibitor of LAR was placed in the inhibitor well.
The assay was performed using the following injection cycle, with the indicated final reagent concentrations in the injection channel: (1) buffer; (2) substrate (17 uM); (3) buffer;
(4) substrate + enzyme (83 nM); (5) buffer; and (6) substrate +
inhibitor (66 ~zM) + enz_irme. The total flux of reagents remained constant during each step of the assay by maintaining a constant overall sum of con:~bined currents from the wells .
The raw fluorescent data from this experiment are shown in Figure 4. The control data, e.g., in the absence of NDSB-195, is shown as a gray line, running at or near the baseline of the data. As can be :peen from this data, LAR action on the dFMUP
substrate produce:> only a moderate signal, ranging between a fluorescent intensity o:E 2200 and 2250. Further, while some effect of the inhibitor is apparent through this assay at later time points, that effects is relatively small. Without being bound to a particular theory, it is believed that this is the result of two phenomena: (I) the hAR enzyme is interacting with the channel walls, resulting i.n a srnearing of the enzyme throughout the assay, as indicated by tree appE:arance of signal in the substrate only control; and ( 2 ) t:he high electrophoretic mobility of the_ dFT2CTP

substrate opposite the electroosmotic mobility of the system results in the substrate and enzyme being separated, thereby reducing the ability of the enzyme to act on the substrate.
Upon inclusion of NDSB-195 in the assay system, however, the data became much clearer (black line). In particular, the inclusion of NDSB in this assay shows dramatic improvements in signal over the same system without the zwitterionic component, including a lack of signal in the substrate only control.
Further, the effects of the inhibitor also are much more dramatic and clearly evident. The assay was run in continual cycle for six hours with no detectable loss of signal or increase in background fluorescence.
All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

We Claim:

1. A method of enhancing material direction and transport by electroosmotic flow of a fluid containing said material, comprising providing an effective concentration of at least one zwitterionic compound in said fluid containing said material.

2. The method of claim 1, wherein said effective concentration of said zwitterionic compound in said fluid is greater than about 5 mM.

3. The method of claim 1, wherein said effective concentration of said zwitterionic compound in said fluid is from about 5 mM to about 2 M.

4. The method of claim 1, wherein said zwitterionic compound is selected from betaine, sulfobetaine, taurine, aminomethanesulfonic acid, a zwitterionic amino acid, HEPES, CAPS, MES, and tricine.

5. The method of claim 4, wherein said zwitterionic compound is a non-detergent sulfobetaine.

6. The method of claim 5, wherein said non-detergent sulfobetaine is selected from dimethylethylaminopropane sulfonic acid, dimethylbenzylaminopropane sulfonic acid, and 3-(N-pyridinium)propane sulfonic acid.

7. The method of claim 1, wherein said material comprises a plurality of differentially charged chemical species.

8. The method of claim 1, wherein said material comprises a protein.

9. The method of claim 8, wherein said protein is an enzyme.

10. The method of claim 1, wherein said direction and transport of a fluid is carried out in at least one microscale channel.

11. The method of claim 1, wherein said fluid containing said material is disposed in a microscale fluidic system which comprises:
a substrate having at least two intersecting channels disposed therein, at least three ports disposed in said substrate and in fluid communication with free termini of said at least two intersecting channels; and a separate electrode placed in electrical contact with each of said port, whereby a fluid contained in each of said ports is in electrical contact with said electrodes.

12. A method of transporting at least first and second discrete fluid volumes along a fluid filled channel by electroosmosis, wherein said at least first discrete fluid volume comprises at least a first chemical species having a different net charge than at least a second chemical species contained in said second discrete fluid volume, the method comprising:
providing an effective concentration of a zwitterionic compound within each of said at least first and second discrete fluid volumes; and applying a voltage from one point in said channel to a different point in said channel whereby said at least first and second fluid volumes are transported along said fluid filled channel.

13. The method of claim 12, wherein said first and second discrete volumes are transported along said fluid filled channel, substantially without intermixing either of said first or second chemical species.

14. The method of claim 13, wherein each of said at least first and second discrete fluid volumes comprise at least two differentially charged chemical species.

15. A method of reducing electrophoretic mobility of charged chemical species in a microscale channel having a fluid disposed therein, and which channel has a voltage applied thereacross, comprising providing an effective concentration of at least one zwitterionic compound in said fluid.

16. The method of claim 15, wherein said charged chemical species is a positively charged chemical species.

17. The method of claim 15, wherein said charged chemical species is a negatively charged chemical species.

18. A microfluidic system comprising:
at leash three ports disposed at free termini of at least two intersecting fluid channels, wherein at least one of said channels has at least one cross-sectional dimension of from about 1 µm to about 500 µm, and wherein at least one of said channels is capable of propagating free electroosmotic flow of a fluid in said channel;
an electrode placed in electrical contact with each of said ports; and at least one fluid disposed in at least one of said channels, whereby said fluid is in electrical contact with said electrodes, and wherein said fluid comprises an effective concentration of zwitterionic compound.

19. The microfluidic system of claim 18, wherein said fluid channels comprise grooves disposed in a surface of a first planar substrate, and a second planar substrate overlays said first planar substrate to form said fluid channels.

20. The microfluidic system of claim 18, wherein at least one of said first and second planar substrates comprise silica.

21. The microfluidic system of claim 18, wherein said system further comprises a fluid direction system which concomitantly and separately modulates a voltage applied at each of at least three of said electrodes.