Surface adsorption of biological materials, such as proteins, to the walls of microscale fluid conduits can cause a variety of problems. For example, in assays relying on flow of material in the conduits, adsorption of test or reagent materials to the walls of the conduits (or to reaction chambers or other microfluidic elements) can cause generally undesirable biasing of assay results.
For example, charged biopolymer compounds can be adsorbed onto the walls of the conduits, creating artifacts such as peak tailing, loss of separation efficiency, poor analyte recovery, poor retention time reproducibility and a variety of other assay biasing phenomena. The adsorption is due, in part, e.g., to electrostatic interactions between, e.g., positively charged residues on the biopolymer and negatively charged groups resident on the surface of the separation device.
Reduction of surface adsorption in microscale applications is typically achieved by coating the surfaces of the relevant microscale element with a material which inhibits adsorption of assay components. For example, glass and other silica-based capillaries utilized in capillary electrophoresis have been modified with a range of coatings intended to prevent the adsorption of charged analytes to the walls of the capillaries. See, for example Huang et al., J. Microcol. Sep. 4, 135-143 (1992), Bruin et al., Journal of Chromatogr., 471, 429-436 (1989); Towns et al., Journal of Chromatogr., 599, 227-237 (1992); Erim, et al., Journal of Chromatogr., 708, 356-361 (1995); Hjerten, J. Chromatogr., 347, 191 (1985); Jorgenson, Trends Anal. Chem. 3, 51 (1984); and McCormick, Anal. Chem., 60, 2322 (1998). These references describe the use of a variety of coatings, including surface derivatization with poly(ethyleneglycol) and poly(ethyleneimine), functionalization of poly(ethyleneglycol)-like epoxy polymers as surface coatings, functionalization with poly(ethyleneimine) and coating with polyacrylamide, polysiloxanes, glyceroglycidoxypropyl coatings and others. Surface coatings have also been used for, e.g., modification of electroosmotic potential of the relevant microscale surface e.g., as taught in U.S. Pat. No. 5,885,470, CONTROLLED FLUID TRANSPORT IN MICROFABRICATED POLYMERIC SUBSTRATES by Parce et al.
Other than the use of surface coatings, few approaches exist for controlling surface adsorption of biopolymers in microscale systems. In general, other design parameters used to control adsorption include the material used in the device, modulation of flow rates and the like. Generally, surface adsorption of biological materials in capillary fluidics applications is a significant issue for at least some applications, and additional mechanisms for inhibiting surface adsorption in microfluidic applications are desirable. The present invention provides new strategies for inhibiting surface adsorption of polymers, molecules and biological materials, e.g., in pressure-based microscale flow applications. Additional features will become apparent upon complete review of the following disclosure.
The present invention derives from the surprising discovery that electroosmotic flow can be,used in a pressure-driven microfluidic system to modulate surface adsorption. In particular, application of an electric field in a fluidic conduit during pressure-based flow prevents or reduces adsorption of materials such as proteins from adhering to the walls of a microchannel or other microscale element. Thus, application of an electric field during pressure-based flow can be used to reduce adsorption of proteins and other molecules or materials to the walls of the microscale element. Thus, application of electrokinetic force during pressure based flow can be regular and reversible, e.g., as applied by an alternating current. In this embodiment, movement of components in a microscale conduit due to electrokinetic forces can be minimized, a desirable feature, e.g., for applications in which separation of materials by charge is not desired.
Accordingly, the invention provides methods of regulating surface adsorption in a channel. In the method, a fluid is flowed through a channel by applying pressure to the fluid in the channel. An electric field (which is alternating or constant) is continuously or periodically applied to, the fluid in the channel. The electric current field can be used as an additional motive force directing movement of a material in the fluid (or directing the fluid itself, as occurs, e.g., during electroosmosis) adding or subtracting from the pressure-based velocity of the material in the channel (electrokinetic and pressure-based flow effects can have the same or an opposite force vector). Alternatively, the electric field can be applied in such a way that the effects of the electric field on the overall velocity of the material are negligible or non-existent (e.g., other than at the walls of the conduit, where the electric field modulates adsorption). For example, the overall contribution to the velocity of a fluid or material in a fluid, exclusive of adsorption effects, can be anywhere from 0.1xc3x97 of the total velocity or less, to less than 50% of the total velocity (0.5xc3x97) to 90% of the total velocity (0.9xc3x97) or more. For example when using alternating current, there may be essentially no contribution to velocity of fluids or materials in the fluids.
A variety of fields and current types can be used in the methods of the invention. For example, an alternating square wave or sine wave field can be applied. Similarly, adsorption of a variety of materials can be regulated by the application of electric fields, including proteins, cells, carbohydrates, nucleic acids, lipids and a combination thereof Application of the electric field can be simultaneous with application of a pressure gradient, or pressure and electrokinetic forces can be alternated. Pressure gradients can be applied by any of a variety of methods, including use of a vacuum source, a hydraulic pressure source, a pneumatic pressure source, an electroosmotic pressure pump, or contact with an absorbent material or a set of fluidly coupled capillary channels.
The use of electrokinetic movement of materials during pressure-induced flow can also be used in conjunction with other methods of eliminating surface adsorption. For example, a coating can be applied to a microscale element to additionally reduce adsorption, or, e.g., to provide for electroosmosis.
In addition to the use of electrical current to prevent adsorption of materials to walls of conduits, adsorption prevention agents can also be used to reduce unwanted adsorption, including, e.g., detergents and blocking agents (e.g., a combination of NDSB and BSA). These adsorption prevention agents can be used in place of or in concert with application of electric fields for reduction of surface adsorption.
Devices and systems for practicing the methods of the invention are also provided. The devices and systems include a body having a one or a plurality of fluidly coupled microchannels disposed therein. A source of fluidic material is fluidly coupled to at least one of the plurality of microchannels. A fluid pressure controller is fluidly coupled to the at least one microchannel and at least-two electrodes are in fluidic or ionic contact with the at least one microchannel. An electrical controller is typically in electrical contact with the at least two electrodes. In a preferred embodiment, the electrical controller applies an alternating electrical field between the at least two electrodes. Typically, the device is configured to apply an electric field of sufficient duration and intensity to dislodge a protein from a surface of the at least one microchannel, or to prevent protein binding to a surface of the at least one microchannel.
In general, the device or system can be configured for electrokinetic or pressure-based flow, or both. For example, flow can be primarily driven by pressure with a small or negligible contribution by electrokinetic forces, or, optionally, the electrokinetic forces can contribute similar or even greater velocity to a material or fluid than the pressure-based forces. In one aspect, the electrical controller is configured to minimize movement of the fluidic material in a direction of fluid flow, or to minimize movement of charged fluidic material in the direction of flow of the charged material. Typically, the fluid pressure controller and the electrical controller concomitantly apply a fluid pressure gradient and an electric field in the at least one channel. Thus, the device or system can include a control element such as a computer with an instruction set for simultaneously regulating electrical current and fluidic pressure in the at least one channel (or any other microscale element in the device). For example, the computer can regulate electrical current, e.g., to control adsorption of biological materials in conduits in the microscale system.
The body of the device or system is typically fabricated from one or more material(s) commonly used in microscale fabrication, including ceramics, glass, silicas, and plastics or other polymer materials. The microscale elements (e.g., microchannels) within the body structure typically have at least one dimension between about 0.1 and 500 microns. Ordinarily, the body has a plurality of intersecting microchannels formed into a channel network.
As noted, the device or system will ordinarily include an alternating current electrical controller e.g., which is capable of generating a sine or square wave field, or other oscillating field.
The device or system will ordinarily include a signal detector mounted proximal to a signal detection region, fluidly coupled to the at least one microchannel. This detector can be configured to monitor any detectable event, e.g., an optical, thermal, potentiometric, radioactive or pH-based signal.
In one aspect, the invention includes an integrated system for moving adherent materials in a microchannel. The integrated system includes a first microchannel fluidly coupled to a pressure source and to a plurality of electrodes. A fluid pressure modulator modulates pressure at the pressure source. An electrical controller modulates current or voltage at least at two of the plurality of electrodes. A computer is operably linked to the fluid pressure modulator and to the electrical controller. The computer has an instruction set for controlling the fluid pressure modulator and the electrical controller. This instruction set provides for reduced adsorption of the adherent materials to a surface of the microchannel during pressure-induced flow of the materials. As noted, the integrated system can include a signal detector proximal to a signal detection zone that is fluidly coupled to the microchannel. The integrated system optionally includes a body structure having the first microchannel disposed therein. The integrated system can include a plurality of additional microchannels disposed in the body structure which are fluidly coupled to the first microchannel. The additional microchannels are fluidly coupled to a source of one or more fluidic reagents, with the computer optionally including an instruction set which directs movement of at least one of the one or more fluidic reagents into the first microchannel.
Similarly, the integrated system can include a flow sensor, which senses the rate of flow of one or more fluidic component in the microchannel. In this embodiment, the computer has an instruction set directing the electrical controller to apply an electric field in response to the rate of flow of the one or more fluidic component.
Definitions
Unless specifically indicated to the contrary, the following definitions supplement those in the art for the terms below.
xe2x80x9cMicrofluidic,xe2x80x9d as used herein, refers to a system or device having fluidic conduits or chambers that are generally fabricated at the micron to submicron scale, e.g., typically having at least one cross-sectional dimension in the range of from about 0.1 xcexcm to about 500 xcexcm. The microfluidic systems of the invention are fabricated from materials that are compatible with the conditions present in the particular experiment of interest. Such conditions include, but are not limited to, pH, temperature, ionic concentration, pressure, and application of electrical fields. The materials of the device are also chosen for their inertness to components of the experiment to be carried out in the device. Such materials include, but are not limited to, glass and other ceramics, quartz, silicon, and polymeric substrates, e.g., plastics, depending on the intended application.
A xe2x80x9cmicroscale cavityxe2x80x9d is a conduit or chamber having at least one dimension between about 0.1 and 500 microns.
A xe2x80x9cmicrochannelxe2x80x9d is a channel having at least one microscale dimension, as noted above. A microchannel optionally connects one or more additional structure for moving or containing fluidic or semi-fluidic (e.g., gel- or polymer solution-entrapped) components.
A xe2x80x9cmicrowell platexe2x80x9d is a substrate comprising a plurality of regions which retain one or more fluidic components.
A xe2x80x9cpipettor channelxe2x80x9d is a channel in which components can be moved from a source to a microscale element such as a second channel or reservoir. The source can be internal or external (or both) to the main body of a microfluidic device comprising the pipettor channel.
An xe2x80x9celectric currentxe2x80x9d is a flow of charge from one place to another. The typical unit of current flow is the ampere. For purposes of this disclosure, an xe2x80x9celectric fieldxe2x80x9d exists wherever an electric or ionic force acts on a charged element (particle, atom, molecule, etc.). In the context of a microscale channel, an electric field of the invention can be, e.g., an electronic or ionic gradient across a selected microchannel region or area. Thus, typically, electrodes are positioned in wells which are fluidly coupled to channel termini. In this configuration, the electrodes are in electrical contact with fluid in the wells (i.e., where electrons travel to or from the fluid into or from the electrode) and in ionic contact with fluid in the channels.
A xe2x80x9cdirect currentxe2x80x9d is typically described in terms of its direction and magnitude. The current flows in a specified direction, with a specified magnitude of flow (e.g., a current between a positive electrode and a negative electrode could be, e.g., 1 milliamp). Classically, the direction of an electric field is the direction of the force on a positive test charge (real or hypothetical) placed at a specified point in the electric field (the force on a negative charge such as an electron is, therefore, opposite the xe2x80x9cdirectionxe2x80x9d of the field). In an xe2x80x9calternating current,xe2x80x9d the current flows back and forth between two or more specified points in a circuit, e.g., in a microfluidic system, two electrodes connected by an fluid comprising ionizable elements. This back and forth flow is typically, though not necessarily, regular. Thus, alternating current (AC) has a frequency (e.g., the number of times the flow goes through a complete cycle per unit time) and an amplitude. The function used to describe the profile of the AC can be essentially any regular repeating wave form. Common wave forms include trigonometric functions (e.g., sine or cosine waves), square waves and an essentially infinite variety of other functions. One of the most common AC forms is sine wave AC, with the variation of the alternating current with time following the formula I=Imax sin 2xcfx80ft where I is the current, Imax is the maximum current, f is the frequency of the current and t is the specified time (typically, I=0 and is increasing, when t=0). By analogy with circular motion, angular frequency xcfx89 (in radians per second) is often used in discussions of sinusoidal alternating currents instead of frequency f (e.g. in hertz), where xcfx89=2xcfx80f and the instantaneous current in an ac circuit is I=Imax sin xcfx89t. For a typical sine wave alternating current, the effective current (Ieff) is       I    eff    =                    I        max                    2              .  
Similarly, for a typical sinusoidal ac, the effective voltage (Veff) is       V    eff    =                    V        max                    2              .  
Both AC and DC can exist as wave functions, e.g., as described above for AC, and where the current is varied between 0 and a selected Imax. For example, a xe2x80x9csquare wavexe2x80x9d can be either DC or AC, or have both DC and AC components. A square wave is characterized by an abrupt wave function in which the transition between Imax and Imin is very rapid (for a perfect square wave, the transition is instantaneous). Both AC and DC functions can be present simultaneously. For example, an AC/DC square wave exists where direct current is turned off and on repeatedly and abruptly. An AC square wave exists where the current oscillates between Imax and Imin with a fast or even essentially instantaneous transition between the two. It should be noted that, for purposes of this disclosure, a xe2x80x9csquare wavexe2x80x9d includes closely related wave forms, i.e., where the transition between Imax and Imin is substantially more abrupt than the transition that occurs for a sine wave current (i.e., where the transition between Imax and Imin is more rapid than for a sine wave current), even if the transition is not actually instantaneous.
Pressure or electrokinetic forces xe2x80x9csignificantlyxe2x80x9d direct flow of a specified material when the force contributes at least 25% of the total forward velocity of the material, or where the force inhibits flow by at least 25% in the direction of flow (e.g., where electrokinetic and pressure based forces have an opposite force vector, e.g., where the pressure and electrokinetic effects are xe2x80x9ccounter currentxe2x80x9d).