WO1998046986A1

WO1998046986A1 - Method for translocating microparticles in a microfabricated device

Info

Publication number: WO1998046986A1
Application number: PCT/US1998/007392
Authority: WO
Inventors: Zhonghui Hugh Fan; Sterling Eduard Mcbride; Satyam Cherukuri
Original assignee: Sarnoff Corporation
Priority date: 1997-04-15
Filing date: 1998-04-14
Publication date: 1998-10-22
Also published as: AU7358698A

Abstract

A method for translocating microparticles in a capillary or microfabricated device comprising pumping a fluid in the capillary or device, wherein channels (101 and 102) included in the capillary or microfabricated device are less than about 500 νm wide and less than about 500 νm deep or are less than 500 νm in diameter. This method provides an ability to conduct sequential reactions on molecules attached to the translocatable microparticles.

Description

METHOD FOR TRANSLOCATING MICROPARTICLES IN A MICROFABRICATED DEVICE

This invention was made with U.S. Government support under Contract No. N66001-96-C-8630. The U.S. Government has certain rights in this invention. Related Applications

This patent application is related to the following co-pending U.S. patent applications: Serial No. 08/556,036, filed November 9, 1995, entitled A PARTITIONED MICROELECTRONIC DEVICE ARRAY (Zanzucchi et al.); Serial No. 08/556,423, filed November 9, 1996, entitled ELECTROKINETIC PUMPLNG (Zanzucchi et al.); Serial No. 08/645,966, filed May 10, 1996, entitled ELECTROKINETIC PUMPING (Zanzucchi et al.); Serial No. 08/483,331, filed June 7, 1995, entitled METHOD AND SYSTEM FOR

LNHLBΓTLNG CROSS-CONTAMLNATION ΓN FLUIDS OF COMBINATORIAL

CHEMISTRY DEVICE (Demers); Serial No. 08/742,317, filed November 1, 1996, entitled ASSAY SYSTEM (Roach et al.); Serial No. 08/745, 66, filed November 8, 1996, entitled FLELD-ASSISTED SEALING (Fan et al.); Serial No. 08/786,956, filed January 22, 1997, entitled PARALLEL REACTION CASSETTE AND ASSOCIATED DEVICES (Southgate et al.); Serial No. 08/742,971, filed November 1, 1996, entitled MAGNET (McBride); Serial No. 08/554,887, filed November 9, 1995, entitled METHOD OF PRODUCING MICRO- ELECTRONIC CONDUITS (Thaler et al.); Serial No. 08/664,780, filed June 14, 1996, entitled AUTOMATED NUCLEIC ACID SOLUTION (Southgate et al.); Serial No. 08/730,636, filed October 1 1, 1996, entitled LIQUID DISTRIBUTION SYSTEM (Demers et al.); and Serial No. 08/821,480, Attorney Docket No. SARNOFF- 12337, filed March 21, 1997, entitled BALANCED ASYMMETRIC ELECTRONIC PULSE PATTERNS FOR OPERATING ELECTRODE-BASED PUMPS (McBride). The aforementioned patent applications shall be referred to hereinbelow by their respective serial numbers only.

The present invention relates to the field of diagnostics, and, in particular, to the conduct of diagnostic procedures in the context of a capillary or a microfabricated device comprised of capillaries or channels, as further set forth hereinbelow. Whether for medical, veterinary, forensic, public health, or agricultural purposes, diagnostic procedures require the application of a test to identify the presence or absence of some molecule that is detectable because of some inherent characteristic of that molecule. Such tests commonly take place on a sample taken from a patient, animal, plant, soil, and the like, wherein the sample has been placed in solution. Typical tests require dissolved samples of at least 0.1 milliliter. Once in solution, the ability of the test to identify the molecule is limited by the sensitivity of the test and the concentration of the molecule in solution.

Obviously, the less volume required for a given test, the less sample and reagents will be consumed. Other advantages of reducing the volume of the test include reduction in analysis time. Further, using lesser volumes may provide an opportunity to provide sample aliquots having greater concentration of the tested molecule, which in turn will increase the sensitivity and reliability of the test. Moreover, lesser volume requirements for the test increase the ability to have multiple aliquots of the sample to be tested, thus providing an ability to repeat a test on a particular sample to assure accuracy.

One approach to uniform testing procedures for reduced volume sample aliquots is to use a microfabricated device composed of reservoirs and channels, as described in Serial Nos. 08/556,036, 08/483,331, and 08/730,636. Such a device is referred to as a "chip". The chip has been shown to be useful in the application of combinatorial chemistry protocols for the development of libraries of molecules that can be subsequently tested for pharmaceutical activity. See the patent applications cited above. In the combinatorial chemistry application, microparticles have been included in the chip, however, the microparticles have not been caused to move outside of the chamber in which they have been placed. For the diagnostic application of the chip referred to above, however, movement of microparticles within the chip, between and amongst the contained reservoirs, would be advantageous.

SUMMARY OF THE INVENTION The present invention provides methods for the use of a capillary or channel for physical, chemical or biological testing or analysis, wherein a microparticle that is translocatable within the capillary or channel is also included. Both capillaries and channels as used in the context of the present invention are conduits for transporting fluids including reagents and particulate matter from one location to another within a particular capillary or microfabricated device. A capillary can have any suitable shape, such as a cylinder, and is attached at its ends to provide fluid communication between the structures to which the ends are attached. The section of the capillary between its ends may or may not be attached to anything, such as a component of a microfluidic device. In contrast, channels, which also provide fluid communication between non-touching structures, are embedded in or constructed from the same material from which the aforementioned structures are made. The capillary can be separate of other structures, or it can be included in a microfabricated device wherein the capillary interconnects at least some of the included reservoirs or channels thereof.

In particular, the present invention relates to a method for translocating microparticles in a microfabricated device, wherein the microfabricated device comprises a fluid, the method comprising pumping the fluid in the microfabricated device so as to translocate the microparticles, wherein the microfabricated device comprises a channel or a capillary, wherein further the channel or capillary is less than about 500 μm in diameter or less than about 500 μm wide and less than about 500 μm deep. Preferably, the device comprises a first chamber and one or more second chambers that are interconnected by the channels or capillary; wherein the first chamber has dimensions of from about 50 μm to about 5 mm wide, from about 50 μm to about 5 mm long, and from about 10 μm to about 300 mm deep. More preferably, the first chamber has dimensions of from about 100 μm to about 1 mm wide, from about 100 μm to about 1 mm long, and from about 20 μm to about 100 μm deep; and the volume capacity of the first chamber is from about 0.1 nl to about 10 μl; more preferably, the first chamber has a volume of from about 0.1 nl to about 1 μl. The device preferably has at least one first or second chamber that has a deformable wall. The capillary preferably has a diameter of from about 2 μm to about 500 μm; more preferably, from about 5 μm to about 250 μm; yet more preferably, from about 10 μm to about 100 μm. The capillary preferably has a length of from about 1 cm to about 100 cm. Preferably, the channels of the microfabricated device have dimensions of from about 15 μm to about 500 μm wide, from about 10 μm to about 500 μm deep, and from about 1 mm to about 500 mm long; more preferably, the channels have dimensions of from about 30 μm to about 300 μm wide, from about 20 μm to about 300 μm deep, and from about 1 mm to about 100 mm long; yet more preferably, the channels have dimensions of from about 30 μm to about 150 μm wide, from about 20 μm to about 100 μm deep, and from about 1 mm to 50 mm long. The channels are preferably situated coplanar with respect to the first chamber; or, alternatively, not coplanar with respect to the first chamber.

In one embodiment of the present invention, the microparticle used in the context of the capillary or microfabricated device is magnetic; preferably, the microparticle is paramagnetic; more preferably, the microparticle is superparamagnetic.

In another embodiment, the microparticle used in the context of the capillary or microfabricated device has a moiety attached thereto. Preferably, the moiety comprises a biological cell or a substructure thereof; alternatively, the moiety comprises an organic, inorganic, or organometallic group. More preferably, the moiety comprises an amino acid, a polypeptide, a nucleotide, a nucleoside, a nucleic acid, a carbohydrate, or an organic compound, or a combination thereof. Yet more preferably, the moiety comprises a molecule that preferentially or, more preferably, exclusively binds to a second molecule. Such a molecule, includes, but is not limited to, avidin, streptavidin, biotin, or fluorenylmethoxycarbonyl (FMOC), an antibody, or a protein that binds to immunoglobulins, such as Protein A, or a lectin.

In yet another embodiment, the device or capillary further comprises one or more magnets for seizing or translocating a microparticle. Preferably, the magnet is a permanent magnetic, such as that of the neodymium-iron-boron class of permanent magnets. Alternatively, the magnet is an electromagnet. Preferably, the magnet is fixed in place, such as fixed adjacent to a channel, the first chamber, a second chamber, or a combination thereof, or at a position adjacent to the capillary. More preferably, the permanent or electromagnetic is integrated into the device by means of conventional micromachining methods. Alternatively, the magnet is movable, with respect to the device or capillary. For example, the magnet can be moved in a device to a location adjacent to a channel, a capillary, the first chamber, a second chamber, or a combination thereof, or to a location that is not adjacent to the device.

The method for translocating the aforementioned microparticle preferably comprises pumping fluid within the microfabricated device. In one embodiment, the pumping is provided by means of electroosmosis, hydrodynamic pressure, electrohydrodynamic pressure, thermal energy, thermopneumatic force, piezoelectric force, or electrostatic force. Preferably, the electroosmosis results from the application of an electric field to the device, wherein the capillary or chambers and channels through or to which the microparticles are pumped are filled with a buffer. More particularly, the electric field is provided by a potential of from about 200 volts to about 30,000 volts, more preferably from about 200 volts to about 10,000 volts, wherein the potential is applied by means of electrodes located at the outside boundaries of chambers or within channels or capillaries between which the pumping is effected. An alternative means for pumping is predicated on having a deformable wall as part of the structure of a first or second chamber, wherein the pumping is effected by a reversible actuator that deforms the wall of a first or second chamber.

In another embodiment, the present invention relates to a method for translocating microparticles in a first capillary or in a channel of a microfabricated device, wherein the first capillary or channel comprises a fluid and the device comprises a first chamber and one or more second chambers that are interconnected by the channel or a second capillary, the method comprising pumping the fluid in the first or second capillary or channel so as to translocate the microparticles, wherein the first or second capillary or channels are less than about 500 μm wide and less than about 500 μm deep or have less than about a 500 μm diameter, and the pumping is provided by means of electroosmosis, hydrodynamic pressure, electrohydrodynamic pressure, thermal energy, thermopneumatic force, piezoelectric force, or electrostatic force.

In yet another embodiment, the present invention relates to a method for translocating microparticles in a capillary, wherein the capillary comprises a fluid, the method comprising pumping the fluid in the capillary so as to translocate the microparticles, wherein the capillary is less than about 500 μm wide and less than about 500 μm in diameter; wherein further the capillary is in fluid communication with two or more reservoirs; wherein further the microparticle is paramagnetic or superparamagnetic. Preferably, the capillary of the present invention is reversibly combined with one or more magnets for translocating or seizing a microparticle, such that the magnet can be moved along the length of the capillary, held stationary at a particular point along the length of the capillary, or removed from being adjacent to the capillary. In this embodiment, the pumping is preferably provided by means of electroosmosis, hydrodynamic pressure, electrohydrodynamic pressure, thermal energy, thermopneumatic force, piezoelectric force, or electrostatic force; more preferably, the electroosmosis results from the application of an electric field to the device, wherein the capillary, chambers and channels through or to which the microparticles are pumped are filled with a buffer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a microfabricated device in top and cross-sectional views. Figure 2 depicts beads captured in a chamber by a magnet.

Figure 3 depicts a block pattern of a pulse protocol for an electrode-based internal pumping system. Figure 4 illustrates a capillary barrier. Figure 5 is an illustration of a microfluidics device that includes the capillary barrier illustrated in Figure 4.

Figures 6A, 6B, 6C, 6D and 6E displays a liquid distribution system having five plate layers, including gas breaks and gas inlets. Figure 7 is a graph of Velocity (mm/s) versus Electric Field (V/cm). Figure 8 is a graph of Velocity (mm/s) versus NaCl Concentration (mM).

DETAILED DESCRIPTION The present invention relates to the use of microparticles in the context of a microfabricated device or a capillary for the purpose of conducting physical, biological or chemical manipulations, separations, measurements or reactions that are part of a diagnostic test. As noted above, the microfabricated device is also referred to herein as a "chip". The chip or microfabricated device used in the context of the present invention is also referred to as a microfluidic device.

A chip comprises reservoirs for containing, without limitation, reagents, reactants, or waste fluid, which reservoirs in certain embodiments are more particularly referred to herein as chambers, yet more particularly as a first chamber if it is a site for a reaction or as a second chamber if it is a site for storage of any of the fluids used in a chip, whether prior or subsequent to that use. Such fluids include, but are not limited to: (1) a buffer, such as Phosphate Buffer Saline (PBS), a standard phosphate buffer including sodium, potassium, or both ions, and the like; (2) an aqueous solution, such as a sodium hydroxide solution and the like; (3) an organic solvent, such as dimethyl sulfoxide and the like; (4) a suspension of microparticles or other particulate matter in any of the aforementioned buffers, solutions, or solvents; or (5) biological samples, such as blood fluids, homogenized tissue samples, and the like which may be included with any of the aforementioned buffers, solutions, solvents, or suspensions. As will be further discussed below, whereas the first chamber is a site at which a reaction can take place, preferably any location in a capillary or a microfluidics device can be used as the site of reaction by capturing in place one or more reactants, such as by immobilizing a reactant on a surface, which surface is seized at a site in a capillary, reservoir, or channel. Preferably, the surface seized is that of a microparticle, as further described below.

Two reservoirs can be situated physically adjacent to each other such that they have a common orifice through which fluid communication can occur, but which, preferably, is reversibly sealed. Alternatively, the reservoirs can be situated physically remote from each other, which are referred to herein as "non-touching reservoirs", an example of which is depicted in Figure 1, wherein the non-touching reservoirs are labeled A, B, and C. Preferably, non-touching reservoirs are in fluid communications with each other via channels or capillaries, as depicted in Figure 1 as structures 101 and 102, and which are further described below. Reservoirs can have any shape, including but not limited to, spheroid, cube, elliptical, and the like.

In particular, the present invention relates to a method for translocating microparticles in a capillary or in a channel of a microfabricated device, wherein the capillary or device comprises a fluid, the method comprising pumping the fluid in the capillary or device so as to translocate the microparticles. The microfabricated device used in the context of the present invention preferably includes channels filled with the fluid and through which the microparticles are translocated, wherein the channels are preferably less than about 500 μm wide and less than about 500 μm deep, and provide fluid communication between chambers of the microfluidic device.

The present invention also relates to a method for translocating microparticles in a capillary, wherein the capillary is a conduit of fluid that provides fluid communication between other structures, such as non-touching reservoirs, or channels. In cross-sectional areas, a capillary can be identical to a channel as herein defined; preferably, capillaries and channels have approximately identical flow volume characteristics, as determined by cross-sectional area of the capillary or channel conduit. The difference is that whereas both capillaries and channels provide fluid communication between non-touching reservoirs, such as first or second chambers of a microfluidic device, or other channels, and thus both are connected to the non-touching structures or other channels at its ends, the channel is embedded in or otherwise is an integral component of the microfluidic device, and the capillary may be unattached to any structure along its medial section between the communicating ends. Preferably, the capillary has a cylindrical shape having a diameter of from about 2 μm to about 500 μm; more preferably from about 5 μm to about 250 μm; yet more preferably, from about 10 μm to about 100 μm. The microfabricated device can be constructed of any suitable material or combination of materials, including but not limited to glass, quartz, silicon, plastic, and the like, wherein a suitable material is substantially rigid at about 25°C up to at least about 40°C, and remains a solid at a temperature of up to about 120°C. In addition to the channels included in the microfabricated device, a preferable device comprises more than one reservoir, such as a first chamber and one or more second chambers, that are interconnected by the channels or capillaries. The first chamber is alternatively referred to as the reaction chamber, however, one of the advantages of the present method is the ability to use any chamber, any channel, or any capillary, or portions thereof, as the site of the reactions needed for a particular diagnostic procedure, as further discussed below. The second chambers are alternatively referred to as supply or waste chambers. The aforementioned material from which the chip is constructed can vary at or about the reservoirs, such as, for example, including at least one deformable wall at a reservoir, preferably a second chamber. In one embodiment, the chip has more than one reservoir that has a deformable wall.

The first chamber preferably has dimensions of from about 25 μm to about 10 mm wide, from about 25 μm to about 10 mm long, and from about 5 μm to about 500 μm deep. More preferably, the first chamber has dimensions of from about 50 μm to about 5 mm wide, from about 50 μm to about 5 mm long, and from about 10 μm to about 300 μm deep. Yet more preferably, the first chamber has dimensions of from about 100 μm to about 1 mm wide, from about 100 μm to about 1 mm long, and from about 20 μm to about 100 μm deep. The volume capacity of the first chamber is preferably from about 0.1 nl to about 50 μl; more preferably, from about 0.1 nl to about 10 μl; yet more preferably from about 0.1 nl to about 1 μl.

The second chambers have any suitable dimensions such that sufficient reagent and waste chambers are thereby provided in the chip for the diagnostic tests for which the chip is designed. In most applications, volume requirements of the second chambers will not exceed about 500 μl each; preferably, second chambers used for waste disposal have a volume capacity of from about 200 μl to about 500 μl, whereas second chambers used for reagent storage have a volume capacity of from about 50 μl to about 250 μl.

The channels included in the chip preferably have dimensions of from about 5 μm to about 500 μm wide, from about 5 μm to about 500 μm deep, and from about 500 μm to about 500 mm long. More preferably, the channels included in the chip preferably have dimensions of from about 15 μm to about 300 μm wide, from about 10 μm to about 300 μm deep, and from about 500 μm to about 100 mm long. Most preferably, the channels have dimensions of from about 30 μm to about 150 μm wide, from about 20 μm to about 100 μm deep, and from about 500 μm to about 50 mm long. As stated above, capillaries used in the context of the present invention can have the same cross-sectional dimensions as channels. The channels can be situated coplanar or not coplanar with respect to the plane that includes the first chamber and is parallel to an outer surface of the chip, preferably an outer surface having the larger dimensions of the outer surfaces of the chip. For example, for one embodiment that has a coplanar arrangement of channels and reservoirs, all of the channels and reservoirs would be aligned in the same plane as one that is parallel with the wall of the chip. In contrast, an alternative embodiment that has a non-coplanar arrangement has a reservoir situated adjacent to one wall of the chip and all or some of the channels situated adjacent to the other wall of the chip, i.e., the channels or some of the channels are situated in different planes than is at least one of the reservoirs. In such an embodiment, the channel would connect to a reservoir by a bend away from a parallel plane with the adjacent wall, bending toward the reservoir.

As noted above, the present invention includes the translocation of microparticles in a chip or a capillary. A microparticle can have any shape, which is preferably spherical and, when spherical, is referred to as a "bead." Preferably, the microparticle has a length or diameter that does not exceed about 500 μm; and more preferably, the length or diameter is less than about 100 μm. In certain preferred embodiments, the microparticles have a maximum dimension of from about 0.5 μm to about 25 μm, and more preferably from about 1 μm to about 10 μm, and even more preferably, about 2 μm to about 5 μm. Beads used in the context of the present invention preferably have diameters that are less than the cross-sectional dimensions of channels or capillaries when passage through the channels or capillaries is preferred. The cross-sectional dimensions, such as the diameter of a cylinder, define the passage tolerance of a capillary or channel. Conversely, when a microparticle is preferably precluded from passage through the channels or capillaries, the microparticle preferably has a diameter that exceeds at least one of the cross-sectional dimensions, i.e., the passage tolerance, of the channels or capillaries, as further noted below.

Microparticles are comprised of any suitable material, the choice of material being guided by its characteristics, which preferably include minimal non-specific adsorptive characteristics, such as polystyrene. In other embodiments, the microparticles are comprised of, for example, glass, cellulose or a cellulose derivative, plastic, such as nylon or polytetrafluoroethylene ("TEFLON"), metal, ceramic and the like, and combinations thereof. One skilled in the art can choose materials having the characteristic of flexibility when the preferred microparticle has a length or a diameter that approximates the cross-sectional value of the capillary or channel in which the microparticle is to be employed, wherein translocation is desirable. Such flexible microparticles, despite having a diameter that is close to the passage tolerance of a capillary or channel, or even greater than the passage tolerance, can "squeeze" through the capillary or channel when caused to move due to, for example, the present invention for translocating microparticles. Conversely, a rigid material is preferred when the microparticle is only slightly larger than the channel or capillary opening and the design of the particular chip requires that the microparticles remain in a particular reservoir.

A preferred microparticle used in the context of the present invention is magnetic. More preferably, the microparticle is paramagnetic. A paramagnetic microparticle can be comprised of, for example, iron dispersed in a polystyrene matrix, and can be obtained, for example, from Dynal (Oslo, Norway). More preferably, the microparticle is superparamagnetic as sold by Dynal (Oslo, Norway) and other commercial manufacturers. A superparamagnetic microparticle differs from a paramagnetic particle by having substantially no remanence or hysteresis. In other words, superparamagnetic microparticles respond to a magnetic field in the same fashion as paramagnetic microparticles, but whereas the paramagnetic particles exhibit some remanence and hysteresis, and therefore tend to clump together after exposure to a magnetic field ceases, superparamagnetic microparticles completely demagnetize when the field is removed, thus allowing the superparamagnetic microparticles to be redispersed without clumping immediately after the field is removed.

The preferred microparticle has a moiety attached thereto. A suitable moiety provides a means for binding the microparticle to defined molecules or cells or other substrates, or provides a means for signalling the presence of the microparticle. For example, one embodiment of the moiety is one that comprises a cell or a substructure thereof. By substructure, it is intended any of a cell's included structures that are separable or isolable from a cell, as is well known in the art. Such substructures include, but are not limited to, the cell or nuclear membrane, endoplasmic reticulum, Golgi bodies, vacuoles, mitochondria, chloroplasts, and the like.

Another embodiment of the moiety comprises an organic or inorganic compound. Preferably, such a compound comprises an amino acid, a polypeptide, a nucleotide, a nucleoside, a nucleic acid, a carbohydrate, or an organic compound, or a combination thereof. More preferably, the moiety is a binding moiety comprising a molecule that preferentially or, yet more preferably, exclusively binds to a second molecule. Such a molecule includes, but is not limited to, avidin, biotin, streptavidin, fluorenylmethoxycarbonyl (FMOC), an antibody, a protein that binds to immunoglobulins, such as Protein A, or a lectin.

While the device is designed to allow the movement of the microparticles by pumping means, which are further discussed below, in certain embodiments and uses thereof it is preferred to hold or seize the microparticles at a certain location, or to move them as a discrete group. A preferred method for doing so includes use of magnetic microparticles, as discussed hereinabove, and requires that the device further comprise one or more magnets. Such magnets are preferably shaped and composed as disclosed in Serial No. 08/742,971, which is incorporated herein by reference. Preferred shapes for such magnets have an acute angle, as in the apex of a pitched roof-shaped object; and preferred compositions include laminated layers wherein the North and South poles of each layer opposes each other. Preferably, the magnet provides a suitable magnetic field, such as that provided by a highly magnetic permanent magnet formed of rare earths, such as those formed of the neodymium-iron-boron class of permanent magnets (available, for example, from Edmund Scientific, Barrington, NJ). Alternatively, the magnet is an electromagnet. Either the permanent magnet or the electromagnet can be micromachined and integrated into the chip using conventional methods, as set forth by Ahn et al., J. Microelectromech. Syst., 5, 151 (1996), for example.

For keeping the microparticles fixed in a given place, the passage between where the microparticles are situated and regions of the device that are in communication with that place can, for example, be narrower than the broadest dimension of the microparticles. For example, a spherical microparticle having a diameter of about 100 μm would be precluded from entering a channel having dimensions that were less than the recited diameter, particularly if the disparity of dimensions were substantial. Accordingly, a first chamber could be constructed having dimensions of 1 mm wide, 1 mm long, and 1 mm deep, containing the aforementioned spherical microparticles, and connected to channels that are substantially less than 100 μm in width and depth. By substantially less, it is preferable that the difference is at least 5%; more preferably, at least 10%; yet more preferably, at least 20%. Such a first chamber would necessarily contain the aforementioned microparticles.

An alternate approach to keeping the microparticles in a fixed position requires the use of magnetic microparticles, preferably paramagnetic microparticles, more preferably superparamagnetic microparticles, and a magnet, wherein the particles are fixed at the position of the magnet. For example, see Figure 2, wherein alternative positions 205 or 206 of a magnet are indicated, either of which can function to seize microparticles in a reservoir. Preferably, the magnet is fixed adjacent to a reservoir in any position that suitably allows seizure of the microparticles. The reservoir can be a first chamber, a second chamber, or a combination thereof. Indeed, the particles can also be seized in a channel or a capillary by the same means, and have the same effect. More preferably, the magnet is movable, such as to a location adjacent to a reservoir, such as a first chamber, a second chamber, or a combination thereof, to a location that is adjacent to a channel or capillary, or to a location that is not adjacent to the device. Thus, the device used in the context of the present invention has the versatility to having microparticles moved within the device or fixed in place, as per the requirements of the test for which the device is designed. As noted above, another method of moving the microparticles from position to position within the microfabricated device is by pumping fluid within the device. Any pumping device of suitable dimensions can be used as an internal pump in the context of the microfluidics device of the invention. Such pumps can include microelectromechanical systems (MEMS), such as reported by Shoji et al., "Fabrication of a Pump for Integrated Chemical Analyzing Systems," Electronics and Communications in Japan, Part 2, 70, 52-59 (1989) or Esashi et al., "Normally closed microvalve and pump fabricated on a Silicon Wafer," Sensors and Actuators, 20, 163-169 (1989) or piezo-electric pumps such as described in Moroney et al., "Ultrasonically Induced Microtransport," Proc. MEMS, 91_., 277-282 (1991). Other suitable pumps work by means of, for example, hydrodynamic pressure, as set forth by Rose and Jorgensen, Analytical Chemistrv, 60, 642-648 (1988); thermal energy, as set forth by Burns et al., Proc. Natl. Acad. Sci. U.S.A., 93, 5556-5561 (1996); thermopneumatic force, as set forth by Shoji and Esashi, Journal of Micromechanics & Microengineering, 4, 147-171 (1994); piezoelectric force, as set forth by Shoji and Esashi, supra; or electrostatic force, as set forth by Shoji and Esashi, supra, using techniques well known in the art. Preferably, the pumps used in the present invention have no moving parts. Such pumps can comprise electrode-based pumps, which are generically referred to herein as electrokinetic pumps. At least two types of such electrode-based pumping has been described, typically under the names "electrohydrodynamic pumping" (EHD) and "electroosmosis" (EO). EHD pumping has been described by Bart et al., "Microfabricated Electrohydrodynamic Pumps," Sensors and Actuators, A21-A23. 193-197 (1990) and Richter et al., "A Micromachined

Electrohydrodynamic Pump," Sensors and Actuators, A29, 159-168 (1991). EO pumps have been described by Dasgupta et al., "Electroosmosis: A Reliable Fluid Propulsion System for

Flow Injection Analysis," Anal. Chem., 66, 1792-1798 (1994) and Rose and Jorgensen, supra.

EO pumping is believed to take advantage of the principle that the surfaces of many solids, including quartz, glass and the like, become charged, negatively or positively, in the presence of ionic materials, such as salts, acids or bases. The charged surfaces will attract oppositely charged counter ions in solutions of suitable conductivity. The application of a voltage to such a solution results in a migration of the counter ions to the oppositely charged electrode, and moves the bulk of the fluid as well. The volume flow rate is proportional to the current, and the volume flow generated in the fluid is also proportional to the applied voltage. Typically, in channels of capillary dimensions, the electrodes effecting flow can be spaced further apart than in EHD pumping, since the electrodes are only involved in applying force, and not, as in EHD, in creating charges on which the force will act. EO pumping is generally perceived as a method appropriate for pumping conductive solutions.

EHD pumps have typically been viewed as suitable for moving fluids of extremely low conductivity, e.g., 10^"14 to 10^"9 S/cm. It has been demonstrated in Ser. No. 08/730,636, the contents of which are incorporated herein by reference, that a broad range of solvents and solutions can be pumped using appropriate solutes than facilitate pumping, using appropriate electrode spacings and geometries, or using appropriate pulsed or d.c. voltages to power the electrodes.

A more preferred method of pumping uses electroosmosis. Movement of fluid within the device results from the application of an electric field to the capillary or device, wherein the capillary, reservoirs and channels through or to which the microparticles are pumped are filled with a fluid that is conductive, and is preferably a buffer. The buffer, necessarily comprising electrolytes and therefore conductive, is preferably a phosphate buffer that includes potassium or sodium or both, or any other buffer that affords similar buffering capacity. Any fluid as defined herein that is conductive can be used in the context of the electrode-based fluid movement described herein. Preferably, the electric field is provided by a potential of from about 100 volts to about 30,000 volts, more preferably of from about 200 volts to about 20,000 volts, yet more preferably of from about 200 volts to about 10,000 volts, even more preferably, of from about 200 volts to about 5,000 volts, wherein the potential is applied by means of electrodes located at the outside boundaries of chambers or within channels or capillaries between which the pumping is effected. Such electrokinetic methods of pumping are further discussed in the aforementioned related applications 08/556,423 and 08/645,966, which are incorporated herein by reference.

It is believed that an electrode-based internal pumping system can best be integrated into the microfluidics device of the invention with flow-rate control at multiple pump sites and with relatively less complex electronics if the pumps are operated by applying pulsed voltages across the electrodes. Figure 3 shows an example of a pulse protocol 310 where the pulse-width 301 of the voltage is τi and the pulse interval 302 is τ₂. Typically, τi is between about 1 μs and about 1 ms, preferably between about 0.1 ms and about 1 ms. Typically, τ₂ is between about 0.1 μs and about 10 ms, preferably between about 1 ms and about 10 ms. The height 303 of the pulsed width 301 is related to the strength of the voltage applied. A pulsed voltage protocol is believed to confer other advantages, including ease of integration into high density electronics, allowing for hundreds of thousands of pumps to be embedded on a wafer-sized device, reductions in the amount of electrolysis that occurs at the electrodes, reductions in thermal convection near the electrodes, and the ability to use simpler drivers.

The pulse protocol can also use pulse wave protocols that are more complex than the block pattern illustrated in Figure 3. For example, a preferred pulse wave protocol includes periodic reversal of the voltage polarity applied to the electrodes of the pump while maintaining a net flow of liquid in a desired direction. In particular, over an operating period of time encompassing at least one polarity cycle, a ratio of either (a) a first voltage-integrated area associated with one polarity to a second voltage-integrated area associated with the other polarity or (b) a charge carried by the current associated with one polarity to a charge carried by the current associated with the other polarity is preferably between about 1:0.5 and 0.5: 1. This preferred pulse wave protocol has been found to correlate with a substantial decrease in bubble formation at the electrodes, which are preferably eliminated or reduced. See Attorney Docket No. SARNOFF 12337.

Electrokinetic pumps used in the context of the microfluidics devices of the present invention can be further refined in their application by using capillary barriers located within the channels or capillaries of a microfluidics device. An example of such a capillary barrier is shown in Figure 4, which is a detail of a portion of a microfluidics device 101 that is illustrated in Figure 5. Figure 4 illustrates a capillary barrier 370, at which a meniscus 371 forms, at the junction between a first channel 222 containing liquid 1 1 and either a second channel 218 or a third vertical channel 390. The meniscus 371 formed at the outlet of the first channel 222 into second channel 218 will tend to inhibit seepage from the first channel 222, such as the seepage that can result from capillary forces. In some embodiments, there are vents (not illustrated) that extend through the feedthrough plate 300 at the tops of the second channel 218 or the third vertical channel 390.

More complex design considerations than were discussed above can, in some cases, affect the design of the capillary barrier. In some cases it is desirable to narrow the sluice formed by second opening 362 or a third opening (not shown) to increase the impedance to flow (i.e., the frictional resistance to flow) as appropriate to arrive at an appropriate flow rate when the internal electrokinetic pump 360 or 361 is activated. The problem that this design alteration can create is that narrower channels can increase capillary forces, thereby limiting the effectiveness of channel breaks.

Thus, in one preferred embodiment, a channel break further includes one or more upwardly oriented sharp edges 369 (not illustrated). More preferably, a channel break includes two or more upwardly oriented sharp edges 369.

In fabricating apparatuses with capillary barriers, care must be taken to assure the alignment of the various small-scaled features. Accordingly, an effort was undertaken to design a capillary barrier that was more forgiving of deviations in alignment. Such a design is reflected in Figure 6A, which shows a liquid distribution having five plate layers, preferably formed of glass, comprising upper top layer 100 A, and lower top layer 100B, upper center layer 1 10A, lower center layer 110B, and bottom layer 120. Between lower center layer 1 10B and bottom layer 120 is seal 101. Liquid flows from feeder channel 116, through alpha vertical channel 125, through distribution channel 122, to capillary break 170, which is formed by opening 164, which opens into cavity 162 formed in lower top plate 100B. When pump 160 is activated, liquid is pushed past the capillary break until it begins to fall into beta vertical channel 118 and thereafter into reaction cell 150. Reaction cell 150 has drain 155. It will be recognized from prior description that there can be several openings 164 forming several capillary breaks 170 that lead into beta vertical channel 118.

It has been found that the reproducibility of pumping can be improved by assuring that the capillary break occurs at the site intended. One way to do this is to "reset" the capillary break by injecting gas pressure from gas-source channel 102 to blow gas through beta vertical channel 1 18 and opening 162 to clear it of any liquid forming unwanted functional capillary breaks. Suitable gas inlets are shown, for example, in Figures 6B, 6C, 6D and 6E. This gas pressure can back up the liquid in, for instance, the distribution channels 122, without detriment. However, the predominant pathway of gas flow is through the beta vertical channel 118, through reaction cell 150, and out drain 155.

The construction of a liquid distribution system having the capillary breaks of this embodiment as shown in Figure 6A is generally according to the methods described above, with some refinements pertaining to the upper top layer 100A and lower top layer 100B. For instance in Figure 6B, the cavities 162 and opening 164 are formed in a plate of relatively larger thickness than the final lower top plate 100B, for instance a thickness of about 8 to about 20 mils. This plate is thinned to become the lower top plate 100B. A gas source channel 102 is formed in the upper top layer 100A. After the two plates 100A and 100B are joined in the manner described above, the combined thickness of the two plates is reduced by lapping. Finally, holes in which the electrodes of pumps 160 are formed are drilled through the joined combination of upper top plate 100A and lower top plate 100B. Other embodiments are shown in Figure 6C, 6D and 6E. For the device in Figure 6E, the plates do not have to be pre-joined before drilling holes for electrodes if layer 100B is sufficiently thick to be self-supporting during the fabrication steps.

Another preferred method of pumping is effected by a reversible actuator or roller that deforms the wall of a reservoir having a deformable wall. The hardware required to form and work such an actuator or roller are well known in the art, and is disclosed in, for example, Shoji et al., Electronics and Communications in Japan, Part 2, 70, 52-59 (1989) or Esashi et al., Sensors and Actuators, 20, 163-169 (1989).

The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. Example 1 This example sets forth the structure of a microfabricated device used in the context of the present invention. Experiments were performed in a microfabricated device consisting of a channel 101 that connects reservoirs A and C and a second channel 102 that connects reservoirs C and B, as shown in the upper drawing of Figure 1. Reservoirs A, B, and C have the same depth as the channel, as shown in the lower drawing of Figure 1 , which is a cross- sectional view; note that reservoir C widens into a square-shaped compartment with channel connections situated at opposing corner positions that are connected to the aforementioned reservoirs A and B. The device was fabricated using photolithography, chemical and laser etching, and binding techniques, as set forth in detail in Fan et al., Anal. Chem., 66, 177-184 (1994) and Serial Nos. 60/010,513, 08/554,887, and 08/745,766. In particular, the device illustrated in Figure 1 was constructed using Corning 7740 glass plates (2' x 2') that were first patterned using photolithography. The plate was then subject to etching in the mixture of hydrofluoric acid, nitric acid, and water, as recited in Fan et al., supra, so that a designed layout of channels and reservoirs was fabricated therein. Another glass plate was drilled using a laser drill (Resonetics Corp., New Hampshire) to make holes for the introduction of solution into the device. The two glass plates were then placed together, with holes on the top plate being aligned with the ends of the channels. Anodic bonding was used to seal the two plates together, as described in Serial No. 08/745,766. Example 2 This example illustrates the use of electroosmosis for pumping fluid in a microfabricated device. Using the device described in Example 1, channels 101 and 102 were filled with a buffer solution consisting of 50 mM tris(hydroxymethyl)-aminomethane and 50 mM boric acid, and reservoir A was loaded with a bead suspension. The bead suspension consisted of superparamagnetic microparticles, i.e., Dynabeads® , 2.8 μm diameter, from Dynal A.S., Oslo, Norway. Reservoir B was filled with the same buffer solution. An electric potential of 500 volts was applied between reservoirs A and B via electrodes inserted therein, respectively. The electrodes in the solutions in reservoirs A and B were platinum wires. The distance between reservoir A and B is 37 mm. In consequence of the applied electric potential, the buffer solution was pumped electroosmotically from reservoir A through channel 101, reservoir C, and channel 102 to reservoir B, although the direction could be reversed depending on the polarity of the electrodes connected to the respective reservoirs. The speed of pumping depended on the strength of the electrical field was applied, the composition and pH of the buffer to which the electrical field was applied, and other conditions, as further discussed below and at Serial Nos. 08/556,423 and 08/645,966.

In particular, electroosmotic velocity (mm/s) was shown to be linearly proportional to the electric field strength. Velocity of the pumped fluid was measured with respect to applied electric field strengths of 50 V/cm to 400 V/cm; the results are shown in Figure 7, wherein the y-axis is labeled Velocity (mm/s) and the x-axis is labeled Electric Field (V/cm). A decrease in electroosmotic pumping velocity was also shown with increasing concentration of electrolytes, i.e., by altering the buffer, the results provided in Figure 8. Figure 8 is labeled Velocity (mm/s) on its y-axis and NaCl Concentration (mM) on its x-axis. For this experiment, lO mM phosphate buffers at pH 7.2 having various NaCl concentrations (range 50 mM to 750 mM) were used in a microfluidics device, with an electric field of 50 V/cm.

Results obtained were in conformity with that reported for capillary electrophoretic systems, i.e., electroosmotic pumping velocity increases linearly with increasing electric field strength, but decreases asymptotically with increasing electrolyte concentration. See Baker, Capillary Electrophoresis. Chapter 2 (John Wiley & Sons: New York, 1995). Example 3

This example illustrates the use of a magnet to seize magnetic microparticles in a reservoir of a microfabricated device. Using a microfabricated device having a channel that was 150 μm wide, 50 μm deep, and 36 mm long, which was of a construction as disclosed in Example 1, in combination with a rectangular neodymium-iron-boron magnet that was placed against the outside of a reservoir, magnetic microparticles were seized magnetically so as to remain in place in the reservoir while surrounding buffer flowed via electroosmotic pumping as set forth in Example 2. The magnet had 1" x 1/8" x VΛ" dimensions and a strength of about 27-35 million gauss oersteds. The chamber was 1 mm wide, 1 mm long, and 50 mm deep, and the average size of the magnetic particles, which were spherical paramagnetic Dynabeads® from Dynal, was 2.8 μm in diameter. The magnet was placed so that an edge of it was in contact approximately at the center of the outside wall thereof, which edge was the North Pole or South Pole. The capture efficiency of the aforementioned magnetic microparticles by the magnet was found qualitatively to depend on the strength of the magnet and the flow rate of the microparticles. More than 95% of the microparticles were estimated to have been seized under the above described conditions.

Further experiments were performed that allowed elucidation of the following parameters that affect the location in a microfabricated device where the microparticles will be seized by an external magnet. When the magnet was placed within at least 3 mm downstream of the reservoir, the beads were captured in the reservoir, as shown in Figure 2. Figure 2 displays two channels 201 and 202 connected to a reservoir 203, wherein microparticles were caused to flow through the named structures. Because an edge 205 or 206 of a magnet was situated downstream of the reservoir 203, the microparticles 204 were captured or seized in the proximate portion of the reservoir relative to the position of the magnet edge 205 or 206. It is believed that the capture of the microparticles was caused by the slower flow rate of the buffer in the reservoir 203 relative to the channels 201 and 202. Because the reservoir 203 is larger than the channels 201 and 202, the flow is lower and the magnetic microparticles are captured more easily in the reservoir than in the channel 201 or 202.

Orientation of the magnet was also found to affect the efficiency of microparticle capture, a result of the variation in the magnetic field strength in response to the directions of the magnet. The experimental results indicate that the seizing efficiency of the magnet, as determined by the estimated percentage of beads being captured, was greatest when the magnet was oriented to deliver the strongest magnetic field in the direction of the chamber.

While this invention has been described with an emphasis upon a preferred embodiment, it will be obvious to those of ordinary skill in the art that variations in the preferred composition and method may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. For example, the rectangular chamber shown and described herein can be shaped differently. Other possible shapes for the chamber which are within the scope of the invention include diamond and circular shaped chambers. Moreover, the chamber can be defined by Y-shaped or T-shaped channel intersections. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for translocating microparticles in a microfabricated device, wherein the microfabricated device comprises a fluid, the method comprising pumping the fluid in the microfabricated device so as to translocate the microparticles, wherein the microfabricated device comprises a channel or a capillary, and wherein the channel or capillary is: (i) less than about 500 ╬╝m wide and less than about 500 ╬╝m deep, or (ii) has less than about a 500 ╬╝m diameter.

2. The method of claim 1 , wherein:

(a) the device comprises a first chamber and one or more second chambers that are interconnected by the channel or capillary; and/or

(b) the volume capacity of the first chamber is from about 0.1 nl to about 10 ml; and/or (c) the microparticle is paramagnetic or superparamagnetic.

3. The method of claim 1 , wherein the microparticle has a moiety attached thereto.

4. The method of claim 3, wherein the moiety comprises: (a) a biological cell or a substructure thereof; or

(b) an organic, inorganic or organometallic group.

5. The method of claim 2, wherein the device further comprises one or more magnets for translocating or seizing a microparticle.

6. The method of claim 5, wherein:

(a) the magnet is fixed adjacent to a channel, a capillary, the first chamber, a second chamber, or a combination thereof; or

(b) the magnet is movable to a location adjacent to a channel, a capillary, the first chamber, a second chamber, or a combination thereof, or to a location that is not adjacent to the device.

7. The method of claim 1, wherein the device has at least one first or second chamber that has a deformable wall, wherein, optionally, the pumping is effected by a reversible actuator that deforms the wall of a first or second chamber.

8. The method of claim 1 , wherein:

(a) the pumping is provided by means of electroosmosis, hydrodynamic pressure, electrohydrodynamic pressure, thermal energy, thermopneumatic force, piezoelectric force, or electrostatic force; and/or

(b) the electroosmosis results from the application of an electric field to the device, wherein the capillary, chambers and channels through or to which the microparticles are pumped are filled with a conductive buffer.

9. A method for translocating microparticles in a first capillary or channel of a microfabricated device, wherein the first capillary or channel comprises a fluid and the device comprises a first chamber and one or more second chambers that are interconnected by the first capillary or channel or a second capillary or channel, the method comprising pumping the fluid in the first and second capillary or channel so as to translocate the microparticles, wherein the first or second capillary or channels are less than about 500 ╬╝m wide and less than about 500 ╬╝m deep or have less than about a 500 ╬╝m diameter, and the pumping is provided by means of electroosmosis, hydrodynamic pressure, electrohydrodynamic pressure, thermal energy, thermopneumatic force, piezoelectric force, or electrostatic force.

10. A method for translocating microparticles in a capillary, wherein the capillary comprises a fluid, the method comprising pumping the fluid in the capillary so as to translocate the microparticles, wherein the capillary is less than about 500 ╬╝m wide and less than about 500 ╬╝m diameter.

11. The method of claim 10, wherein the capillary is in fluid communication with one or more reservoirs.